1

Snakebite envenoming

José María Gutiérrez1, Juan J. Calvete2, Abdulrazaq G. Habib3, Robert A. Harrison4,

David J. Williams5,6 and David A. Warrell7

1Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, PO Box

11501-2060, San José, Costa Rica

2Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas

(CSIC), Valencia, Spain

3College of Health Sciences, Bayero University, Kano, Nigeria

4Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, UK

5Charles Campbell Toxinology Centre, School of Medicine & Health Sciences, University of

Papua New Guinea, Boroko, National Capital District, Papua New Guinea

6Australian Venom Research Unit, Department of Pharmacology and Therapeutics,

University of Melbourne, Parkville, Victoria, Australia

7Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford,

Oxford, UK

Correspondence to:

J.M.G.

jose.gutierrez@ucr.ac.cr

2

Abstract | Snakebite envenoming is a neglected tropical disease that kills >100,000 and

maims >400,000 people every year. Impoverished populations living in the rural tropics

are particularly vulnerable; snakebite envenoming perpetuates the cycle of poverty. Snake

venoms are complex mixtures of proteins that exert a wide spectrum of toxic actions. The

high variability in snake venom composition is responsible for a plethora of clinical

manifestations in envenomings, ranging from local tissue damage to potentially life-

threatening systemic effects. Intravenous administration of antivenom is the only specific

treatment to counteract envenoming. In addition, analgesics, ventilator support, fluid

therapy, haemodialysis and antibiotic therapy are used. Novel therapeutic alternatives

based on recombinant antibody technologies and new toxin inhibitors are being explored.

Confronting snakebite envenoming at a global level demands the implementation of an

integrated intervention strategy involving the WHO, the research community, antivenom

manufacturers, regulatory agencies, national and regional health authorities, professional

health organizations, international funding agencies, advocacy groups and civil society

insititutions.

[H1] Introduction

Snakebite envenoming is a neglected tropical disease resulting from the injection

of a highly specialized toxic secretion — venom — by a venomous snake into humans,

usually under accidental circumstances. Venom is injected through the snake’s fangs,

which are modified teeth connected via a duct to a venom gland (Fig. 1A). The

composition of snake venoms shows high complexity and diversity1, resulting in a variable

biochemical and toxicological profile that determines a wide range of clinical

3

manifestations. Some toxins in venoms provoke local tissue damage, often resulting in

permanent sequelae, whereas others induce systemic effects, including neurotoxic

manifestations (leading to, for example, respiratory paralysis), bleeding, acute kidney

injury, rhabdomyolysis (generalized breakdown of muscle fibres), cardiotoxicity,

autonomic hyperactivity or thrombosis. Venoms from snakes of the family Viperidae

(viperids) cause local effects and systemic manifestations associated with bleeding,

coagulopathies and hypovolaemic shock2. Venoms from snakes of the family Elapidae

(elapids) predominantly induce neurotoxic manifestations, such as neuromuscular

paralysis2.

The superfamily Colubroidea — or advanced snakes — consists of >2,500 species

with a wide geographical distribution and an extended evolutionary history. The

superfamily includes all venomous snakes classified in the taxon Caenophidia, order

Squamata, suborder Serpentes. The most dangerous species are classified within the

families Viperidae (true vipers and pit vipers) and Elapidae (elapids, for example, cobras,

kraits, mambas and sea snakes) (Fig. 1b–i)3,4. Additionally, some species of the families

Lamprophiidae (lamprophiids; subfamily Atractaspidinae, for example burrowing asps or

stiletto snakes) and several subfamilies of non-front-fanged colubroid snakes are also

capable of inflicting envenomings2.

Because snakes are ectothermic, they are abundant in warmer climates, restricting

the hyperendemic regions for snakebites mostly to tropical countries of the developing

world (especially to Africa, Asia, Latin America and Oceania)5–7. In those countries, contact

between snakes and humans is relatively common particularly in the rainy season when

human agricultural activity coincides with the snakes’ breeding season. Epidemiological

evidence gathered from hospital records underscores the high burden of snakebite

envenomings, which is considerable in terms of mortality and sequelae5,8. Evidence from

community-based surveys in some countries suggests that the actual toll is even higher

than estimates from hospital-based statistics (see for example6). By contrast, inhabitants

of higher-income countries of North America and especially Europe have far less exposure

to venomous snakes and are generally unaware of the scale of the public health problem

4

posed by snakebites elsewhere. Consequently, snakebite envenoming have historically

received little attention from funding bodies, public health authorities, the pharmaceutical

industry and health advocacy groups, thereby impairing the development of effective

interventions to reduce the social impact of snakebites9,10.

This Primer summarizes the main issues of snakebite envenoming, including the

epidemiology, the composition of snake venoms, and the pathophysiology, clinical

manifestations, prevention and clinical management of snakebite envenomings. The

global efforts being carried out for reducing the impact of this pathology are described,

together with future trends to better understand and confront this neglected tropical

disease.

[H1] Epidemiology

Snakebite envenoming is a major public health problem in the developing world

(Fig. 2). It is an important cause of morbidity and mortality especially in the impoverished

areas of the warmer tropics and subtropics, such as sub-Saharan Africa, South to South-

East Asia, Papua New Guinea and Latin America6,7,11. Snakebite envenoming occurs in at

least 1.8–2.7 million people worldwide per year, with combined upper estimates of

mortality ranging from 81,410 to 137,880 (Refs5,12). At least 46,000 of these deaths occur

in India alone6. In sub-Saharan Africa, where data are fragmented, mortality estimates

range from 7,000 to 32,000 deaths per year5,7,12, but are likely be underestimated given

that, in West Africa alone, annual mortality has been estimated at 3,557–5,450 (Ref.13).

Snakebites disproportionately affect lower socioeconomic segments of society,

people with poorly constructed housing and those with limited access to education and

health care11,13. Countries with low gross domestic product, Human Development Index (a

composite statistic of life expectancy, education and income) and low healthcare

expenditure are the most affected11. The disease pushes poor people further into poverty

by virtue of high treatment costs, enforced borrowing and loss of income11. Indeed, bites

and fatalities are most common in people 10–40 years of age, comprising the most

productive members of rural communities14. Those <5 years of age experience higher

case-fatality15. In India, the proportion of all deaths from snakebites was highest at ages

5

5–14 years6. Children are exposed while helping in agricultural duties, playing or placing

their hands in rodents’ burrows.

Snakebite envenoming is an occupational and environmental disease of the young

and agricultural workers. The specific populations at risk differ between countries. For

example, tea pickers are at risk in southern India and Sri Lanka, rubber tappers in Liberia,

Thailand, Malaysia, other South East Asian countries and Brazil, and sugar cane workers in

South Africa, Saint Lucia and Martinique16–19. In Myanmar, snakebite envenoming is the

5th leading cause of death, particularly affecting rice paddy farmers20. Fishermen using

hand nets and lines in warmer tropical seas are also at risk, as are families of agricultural

workers. Pregnant women are a highly vulnerable group, and snakebite has been

recognized in Nigeria and Sri Lanka as an important cause of abortion and antepartum

haemorrhage, as well as maternal and fetal loss21.

Snakebite is also an environmental hazard of indigenous nomadic peoples, hunter-

gatherers, tribes, firewood collectors and indigents (extremely poor individuals). This risk

has been documented in South America19, Africa (for example, the Hadza hunter-

gatherers of Tanzania, the African Bushmen of the Kalahari desert or Southern Africa, the

nomadic Fulani and Turkana pastoralists (sheep or cattle farmers) of the savanna in West

Africa and Kenya)22, India and Sri Lanka. Envenoming has been an important cause of

death among the indigenous communities of Australia, of the coastal lowlands of New

Guinea23 and of the Amazon (Yanomami and Waorani ethnic groups)18,19.

A proportion of individuals develop chronic morbidity, disability and psychological

sequelae following snakebite envenoming, including amputations, post-traumatic stress

disorder, blindness, maternal and fetal loss, contractures (permanent shortening of a

muscle or joint often leading to deformity or rigidity), chronic infections and malignant

ulcers7,21,24–26. At least 6,000 amputations owing to snake bites occur annually in sub-

Saharan Africa alone7. Even when chronic disability is not factored in, the burden of

premature death alone as a consequence of snakebite envenoming in India is estimated at

2.97 million disability-adjusted life years, whereas the global burden is conservatively

estimated at 6.07 million disability-adjusted life years6,12,13. This is more than twice the

6

estimate suggested for contributions grouped under ‘venomous animal contact’ in the

Global Burden of Disease study27, underscoring a notorious underreporting of snakebites

in official records. When both premature deaths and disability from snakebites are

factored for 16 countries in West Africa, the combined burden surpasses the worldwide

burdens for other neglected tropical diseases such as buruli ulcer, echinococcosis, leprosy,

trachoma, yaws, yellow fever and podoconiosis. The burden of snakebites in sub-Saharan

Africa is also higher than the burden of trypanosomiasis, leishmaniasis and

onchocerciasis13.

[H1] Mechanisms/pathophysiology

[H2] Snake venom

[H3] Evolution of snake venoms. Venoms are used for predatory and defensive purposes

and have evolved independently in a wide phylogenetic range of organisms, including

snakes, spiders, scorpions and jellyfish28. Venom represents a trophic adaptive trait crucial

for the foraging success of venomous snakes, and as such has had key roles in the

organismal ecology and evolution of advanced snakes29. As a result of rapid evolution

under Darwinian positive selection, venoms comprise protein mixtures of varying

complexity that act individually or as an integrated phenotype to injure or kill the prey or

accidental victim. Despite being traits of moderate genetic complexity in terms of number

of genes that encode toxins, within-species and between-species venom variability seems

to be a common feature at all taxonomic levels30. The mechanisms that generated such

biodiversity remain largely elusive, although genomic reorganizations and other

alterations in genes encoding toxins and toxin expression patterns might be involved31–35.

Like eyes, fins and wings, which have evolved independently in a number of different

lineages, animal venoms have also been shaped by convergent structural and functional

evolution36. Convergent evolution has repeatedly selected a restricted set of genes

encoding proteins containing specific structural motifs as templates for

neofunctionalization (the process by which a gene acquires a new function after a gene

7

duplication event) as venom toxins in different taxa37. Gene duplication, evolutionary

divergence and post-translational quaternary associations in homomer or and heteromer

multiprotein complexes38 add to the complexity of snake venoms, by generating mixtures

of proteins belonging to a handful of multigenic protein families; some of these protein

families exhibit remarkable intrafamily variability39.

Unveiling the spatial and temporal distribution of variations in venom composition

within and between species is essential to understand the evolutionary processes and the

ecological constraints that molded snake venoms to their present-day variability and to

define the phylogeographic boundaries of species. Insights into the selective pressures

that resulted in local adaptation and species-level divergence in venoms can shed light on

the mutual relationship between evolutionary and clinical toxinology: toxins bearing the

highest prey incapacitation activity are often also the most medically important molecules

in the context of a human envenoming. Thus, identifying the molecular basis of venomous

snake adaptation to their natural ecosystems may assist in the identification of those

toxins that must be neutralized to reverse the effects of venom, thereby guiding the

rational development of next-generation snakebite therapeutics40.

[H3] Analysis of snake venom composition. The growing interest in different aspects of

venom biology has catalysed the development of ‘omics’ methodologies aimed at the

qualitative and quantitative characterization of venom toxins, including the proteomics of

venoms (that is, venomics; Box 1). In particular, the combination of next-generation

transcriptomics41,42 and proteomics workflows has demonstrated unparalleled capabilities

for venom characterization in unprecedented detail. Figure 3 summarizes the relative

distribution of the main types of toxic components in viperid and elapid venoms.

A straightforward translational application of the body of knowledge gained

through venomics is the analysis of the immune reactivity of antivenoms against venoms,

a field coined ‘antivenomics’ (Fig. 4). Antivenomics is a proteomics-based protocol to

quantify the extent of cross-reactivity of antivenoms against homologous and

8

heterologous venoms43. The combination of antivenomics and in vivo neutralization tests

constitute a powerful toolbox for evaluating the preclinical efficacy of an antivenom44.

[H2] Mechanism of action

Snakes inject their venoms through a specialized delivery system, which includes a

set of fangs located in the frontal region of the maxillary bones in viperids (Fig. 1a), elapids

and lamprophiids, whereas fangs have a posterior location in non-front-fanged colubroids.

Depending on the size of the fangs, venom is injected either subcutaneously or

intramuscularly. Once delivered, some venom toxins exert local pathological effects in

neighboring tissues, whereas others are distributed systemically through the lymphatic

system and blood vessels, enabling toxins to act at various organs2. Activities of main toxin

families are shown in Box 2, whereas the potential consequences of snakebite

envenoming are shown in Figure 5.

[H3] Local tissue damage. Most viperid and some elapid venoms induce local tissue

damage. Myonecrosis is primarily due to the action of myotoxic phospholipases A2 (PLA2s)

present in these venoms, which bind and disrupt the integrity of the plasma membrane of

muscle fibres45,46. For some PLA2s, disruption of plasma membranes is secondary to

hydrolysis of membrane phospholipids, whereas in the case of catalytically-inactive PLA2

homologues, sarcolemmal damage occurs through hydrophobic interactions47. Calcium

influx into the cytosol occurs following membrane perturbation, causing myofilament

hypercontraction, mitochondrial dysfunction and other degenerative events, leading to

irreversible muscle cell damage46,47. Small basic myotoxic peptides present in some

rattlesnake venoms also induce muscle contracture and can cause necrosis2. Muscle fibers

are also affected by ischaemia resulting from vascular alterations and from increased

pressure in muscles as a consequence of oedema48. Skeletal muscle regeneration requires

the removal of necrotic debris by phagocytic cells, and depends on an intact blood supply

9

and innervation to be successful. In viperid venoms, which affect muscle fibres and

damage the vasculature and nerves, skeletal muscle regeneration is impaired, often

resulting in permanent sequelae48,49.

In addition to myonecrosis, blood vessel integrity is also affected. Snake venom

metalloproteinases (SVMPs) in viperid venoms hydrolyse key components of the

basement membrane of capillaries, particularly type IV collagen, causing the weakening of

the mechanical stability of microvessels. As a consequence, the haemodynamic

biophysical forces operating in the circulation cause distension and, eventually, disruption

of the capillary wall, resulting in extravasation50. SVMP-induced microvascular damage can

also be a consequence of the disruption of endothelial cell-cell adhesions51. SVMPs and

hyaluronidases hydrolyse extracellular matrix components, including various types of

collagens, hyaluronic acid and proteoglycans, affecting the structure and function of not

only microvessels but also other tissue components, thereby playing a part in venom-

induced local tissue damage52.

SVMPs also induce skin damage by degrading the dermal-epidermal interphase,

with the consequent formation of blisters53. Some cobra (Naja sp., family Elapidae)

venoms induce extensive cutaneous necrosis owing to the action of cytotoxins of the

three-finger toxin family54, so named for having a common structure of three loops

extending from a central core, which destabilize plasma membranes of various cell types

in different tissues through a non-enzymatic mechanism55. Locally-acting toxins (that is,

PLA2s and SVMPs) also affect intramuscular nerves49 and other vascular components such

as lymphatic vessels, arterioles and venules56.

An extensive local inflammatory process develops in envenomed tissue, with the

synthesis and release of eicosanoids, nitric oxide, bradykinin, complement anaphylatoxins,

histamine, cytokines, the activation of resident macrophages and other cell types, and the

recruitment of leucocytes57. This inflammatory milieu induces an increased vascular

permeability with formation of an exudate that, in addition to plasma proteins, contains

intracellular and extracellular protein fragments, chemokines, cytokines and damage-

10

associated molecular patterns (DAMPs), which are likely to potentiate the inflammation

and, possibly, contribute to further tissue damage58.

Some mediators also induce pain57. Catalytically-inactive PLA2 homologues excite

pain-related sensory neurons via ATP release and activation of purinergic receptors59.

[H3] Neurotoxicity. Snake venoms of most elapid species, and of some viperid species,

contain neurotoxins that induce a descending flaccid neuromuscular paralysis, which can

involve the life-threatening blockade of bulbar (muscles of the mouth and throat

responsible for speech and swallowing) and respiratory muscles. Two main types of

neurotoxins are found in snake venoms: α-neurotoxins and β-neurotoxins.

α-Neurotoxins belong to the three-finger toxin family and exert their action post-

synaptically at neuromuscular junctions60. They bind with high affinity to the cholinergic

receptor at the motor end plate in muscle fibres, thereby inhibiting the binding of

acetylcholine and provoking flaccid paralysis60.

By contrast, β-neurotoxins are typically PLA2s that act at the presynaptic nerve

terminus of neuromuscular junctions61. For example, the receptor for β-bungarotoxin,

from the krait Bungarus multicinctus (family Elapidae), is the voltage-gated potassium

channel62. Upon binding to their targets, neurotoxic PLA2s induce enzymatic hydrolysis of

phospholipids at the nerve terminal plasma membrane, which causes neurotoxicity63.

Indeed, the generation of lysophospholipids and fatty acids in the membrane cause

biophysical changes that lead to the fusion of synaptic vesicles to the membrane and the

exocytosis of the ready-to-release pool of vesicles46. Furthermore, membrane

permeability to ions is increased, with the consequent depolarization and influx of

calcium, resulting in exocytosis of the reserve pool of vesicles61. Consequently, presynaptic

vesicles are depleted, and intracellular degenerative events ensue, including

mitochondrial alterations, ending up in the destruction of nerve terminals64,65. These

events explain the prolonged and severe paralysis observed in patients. Some neurotoxic

11

PLA2s can also act intracellurlarly after entering the cytosol by endocytosis or through the

damaged plasma membrane62. Within the nerve terminus, PLA2s cause further

degenerative events in mitochondria66.

Other neurotoxins are dendrotoxins and fasciculins, present in the venoms of the

African elapids mambas (Dendroaspis sp.; family Elapidae). Dendrotoxins block voltage-

gated potassium channels at the presynaptic nerve terminal67. Fasciculins, which also

belong to the three-finger toxin family, are inhibitors of acetylcholinesterase68. Their

combined action results in excitatory effects and fasciculations (involuntary muscle

contraction). Some cysteine-rich secretory proteins (CRISPs) in venoms induce paralysis of

smooth muscle2.

[H3] Cardiovascular and haemostatic disturbances. Systemic haemorrhage occurs in

envenomings by viperids and by some species of non-front-fanged colubroids, and can

also develop in envenomings by Australian elapids. In viperid venoms, the main toxins

responsible for systemic haemorrhage are SVMPs, especially those of the PIII class. These

toxins have a multi-domain structure containing exosites (molecular sites distinct from the

active catalytic site that serve as secondary binding sites) that enable them to target the

microvasculature50,69. Bleeding can occur in different organs with several

pathophysiological consequences. For example, intracranial haemorrhage has been

described in envenomings, causing ischaemia and stroke, and leading to neurological

sequelae70,71. The mechanism of action of systemically-acting haemorrhagic SVMPs is

likely to be similar to that described for local haemorrhage, that is, cleavage of key

substrates at the basement membrane of capillaries and at cell–cell junctions, resulting in

the mechanical weakening of the microvessel wall and extravasation50,51.

Snake venoms affect haemostasis in various ways. Many viperid venoms, and some

elapid and non-front-fanged colubroid venoms, contain enzymes that promote

coagulation; these enzymes are either SVMPs or snake venom serine proteinases that act

in the coagulation cascade, such as thrombin-like enzymes or activators of coagulation

factor V, factor X or prothrombin72,73. Some venom enzymes also hydrolyse fibrinogen and

12

fibrin73. SVMPs also release tissue factor74 and affect endothelial function in various ways.

Although these procoagulant components can cause intravascular coagulation, in the

majority of cases they induce a consumption coagulopathy, resulting in defibrinogenation

and incoagulability that are reflected in the alteration of blood clotting tests70. This

condition may contribute to systemic bleeding, especially in venoms containing

haemorrhagic toxins that disrupt the integrity of blood vessels2,70. Some Australian elapid

venoms, which lack haemorrhagic SVMPs but cause coagulopathy secondary to the action

of serine proteinase prothrombin activators, often induce systemic bleeding70.

Many snake venoms affect platelets. SVMP-mediated microvascular damage and C-

type lectin-like proteins contribute to the drop in platelet numbers75. Moreover,

disintegrins, C-type lectin-like proteins, snake venom serine proteinases and some SVMPs

impair platelet aggregation by blocking platelet receptors or by interacting with von

Willebrand factor73,76,77. Thrombocytopenia has been associated with an increased risk of

systemic bleeding in envenomings by haemorrhagic venoms. By contrast, the venoms of

two endemic Caribbean viperid species induce severe thrombosis leading to infarcts in

lungs, brain and heart, despite not being directly procoagulant78. Thrombosis is likely

dependent on SVMP-induced systemic endothelial dysfunction. Acute pituitary

insufficiency secondary to thrombi formation and focal haemorrhage in the anterior

pituitary glands occurs in bites by some viperids79.

Venom-induced systemic bleeding is one of the leading causes of the

haemodynamic disturbances experienced by patients envenomed by viperids, which may

progress into cardiovascular shock2. In these envenomings, hypovolaemia also results

from an increase in vascular permeability, including systemic plasma leakage. This effect is

induced by snake venom serine proteinases that release bradykinin and also by the action

of a plethora of vasoactive endogenous inflammatory mediators. In addition, viperid

venoms contain bradykinin-potentiating peptides, some of which inhibit the angiotensin-

converting enzyme and contribute to haemodynamic alterations80.

13

Hyponatremia, possibly caused by venom natriuretic factor, may play a part in

cardiovascular disturbances in some envenomings81. A direct cardiotoxic effect might also

add to the multifactorial setting of haemodynamic disturbances; sarafotoxins are

responsible for cardiotoxicity in atractaspid venoms2. Sepsis has been described in

snakebite victims as a consequence of infection, further contributing to cardiovascular

dysfunction and shock.

[H3] Acute kidney injury. Some viperid and some elapid snakebite envenomings can lead

to acute kidney injury82. Depending on the type of venom, the following mechanisms have

been associated with the pathogenesis of renal damage: ischaemia secondary to

decreased renal blood flow resulting from the haemodynamic alterations caused by

systemic bleeding and vascular leakage; proteolytic degradation of the glomerular

basement membrane by SVMPs; deposition of microthrombi in the renal microvasculature

(that is, thrombotic microangiopathy), which might also cause haemolysis; direct cytotoxic

action of venom components, such as cytotoxic PLA2s, in renal tubular cells; in the cases of

venoms inducing systemic myotoxicity, accumulation of large amounts of myoglobin in

renal tubules, with consequent toxicity82,83.

[H3] Rhabdomyolysis. Envenomings by sea snakes, some Australian terrestrial elapids,

and some viperid species are associated with systemic myotoxicity, that is,

rhabdomyolysis2. This effect is due to the action of myotoxic PLA2s at the systemic level

owing to the binding of these toxins to receptors in muscle fibers. Myotoxins disrupt the

integrity of the plasma membrane of muscle cells, as described for locally-acting

myotoxins, causing calcium influx and cellular degeneration47. Thus, large amounts of

muscle cytosolic proteins, such as creatine kinase and myoglobin, are released. Deposition

of myoglobin in the renal tubules may contribute to acute kidney injury82.

[H1] Diagnosis, screening and prevention

Snakebites are emergencies that are clinically challenging owing to their potentially

rapid lethality. Uncertainties about species identity and the quantity of venom injected

and its composition, which can vary with the snake’s age and, within species, throughout

14

its geographical range, complicate decision making84. Most snakebites are managed by

nurses or health assistants in district and rural hospitals, clinics, dispensaries and health

posts. In some cases, referral to a provincial tertiary hospital with specialists, intensive

care units and laboratories might be possible.

[H2] Clinical presentation

Victims of snakebite envenoming present with local and systemic symptoms of

envenoming, as well as anxiety and symptoms associated with the treatment they

received before arriving in hospital or health care post. Fear can cause misleading

symptoms such as vomiting, sweating, tachycardia, acroparaesthesiae (abnormal

sensation in extremities), carpopedal spasm (tetany causing painful cramps of hands,

wrists and feet), tachypnoea and hyperventilation leading to syncope, and functional

neurological disorders. Widely practiced traditional first-aid treatments for snakebites

include tight bands or tourniquets85, local incisions, ingestion of emetic herbs or topical

application of herbs, or application of ice, suction, fire or electric shocks at the site of bite.

Not only is the effectiveness of these traditional treatments not proven, but they can

result in treatment-associated comorbidities even in the absence of envenoming such as a

painful, swollen, ischaemic or even gangrenous limb, bleeding or infections.

The specific clinical manifestations associated with bites of viperids, elapids or non-

front-fanged colubroid snakes are described below.

[H3] Viperids. The classic syndrome associated with bites from viperids (including

Viperinae (true Old World vipers and adders) and Crotalinae (Asian pit-vipers, mamushis,

habus and New World rattlesnakes, moccasins, bushmasters and lanceheads)) consists of

local and systemic effects. Local effects in the bitten limb include immediate radiating

pain; rapidly-extending tender swelling with hot inflammatory erythema; signs of

lymphangitis (inflammation of the lymph vessels presenting with red lines on the skin),

which usually becomes evident within 2 hours of the bite; prolonged bleeding from fang

puncture wounds; bullae (blistering); ecchymosis (bruising); tender regional lymph node

enlargement; superficial soft tissue and muscle necrosis and secondary infection (cellulitis

15

or abscess) (Fig. 6a,b)19,86,87. Systemic effects include early syncope and collapse with

transient loss of vision and consciousness; hypotension and shock; cardiac

tachyarrhythmia or bradyarrhythmia; severe bleeding diathesis, spontaneous systemic

bleeding from nose, gums (Fig. 6c), respiratory, gastrointestinal and genitourinary tracts

and sites of recent trauma or healing wounds, and subarachnoid, cerebral, and

antepartum or postpartum haemorrhages; and abortion and fetal death19,86,87.

Variant syndromes consist of symptoms in addition to those described above and are

associated with envenoming by particular species. These symptoms include early

‘anaphylactic’ (autonomic) symptoms (for example, urticaria, angioedema, shock,

sweating, vomiting and diarrhoea), acute kidney injury, generalized increase in capillary

permeability, for example chemosis (Fig. 6d), acute or chronic pituitary failure,

neuromyotoxicity and in situ arterial thrombosis (causing ischaemic infarcts in brain,

kidney, lungs, heart or elsewhere). Envenoming by some rattlesnakes (Crotalus sp.) causes

neuromyotoxic symptoms, characterized by fasciculations in North America and elapid-

like descending paralysis with rhabdomyolysis and acute kidney injury in South America19.

Envenoming by bushmasters (Lachesis sp.) can cause dramatic early autonomic symptoms,

sometimes with vaso-vagal features, and severe local envenoming19. A review of the

species causing these symptoms is beyond the scope of this Primer, but additional

information is provided in Supplementary file S1 and reviewed elsewhere19,86,87.

[H3] Elapids. Bites of elapid snakes (including cobras, kraits, mambas, coral snakes,

Australian and Oceanic venomous snakes, and seasnakes) are associated with the classic

neurotoxic syndrome, which is characterized by flaccid paralysis that is first evident as

bilateral ptosis and external ophthalmoplegia (Fig. 6e,f) sometimes with dilated pupils.

Drowsiness is occasionally mentioned but snake venom toxins are not thought to cross the

blood–brain barrier, so this finding is difficult to explain. Paralysis descends to involve

muscles innervated by lower cranial nerves and neck flexors, as well as bulbar, respiratory,

trunk and limb muscles. Other symptoms are pooling of secretions in the pharynx, loss of

the gag reflex, dyspnoea, declining ventilatory capacity, paradoxical abdominal

respiration, use of accessory muscles and cyanosis, which are ominous signs of impending

16

bulbar and respiratory paralysis. Paralysis is sometimes reversible following treatment

with acethylcholinesterase inhibitors or specific antivenoms, and recovers over time in all

cases, provided that respiration is adequately supported19,23,86–89. Local symptoms include

absent-to-moderate pain, paraesthesiae and local swelling, without blistering or necrosis.

Variant syndromes consist of severe local envenoming with immediate radiating

pain and rapidly extending tender swelling, blistering, superficial, patchy (presence of ‘skip

lesions’) soft tissue necrosis (Fig. 6g) and secondary infection, tender regional lymph node

enlargement, autonomic overactivity and fasciculations, severe abdominal pain

resembling renal or biliary colic (which gradually increases in intensity), excruciating pain

radiating up the bitten limb and acute kidney injury associated with rhabdomyolysis,

hyponatraemia, spontaneous bleeding and coagulopathy, and microangiopathic

haemolysis. Venom spat into the eyes by spitting elapids can lead to ophtalmia, resulting

in intensely painful chemical conjunctivitis with lacrimation and swelling of eyelids, risk of

corneal ulceration, anterior uveitis, and secondary infection leading to permanent

blindness (Supplementary file S1) 19,86,87.

[H3] Non-front-fanged colubroids. Bites from non-front-fanged colubroid snakes

(including the African boomslang (Dispholidus typus), vine snakes (Thelotornis sp.), Asian

keel-backs (Rhabdophis sp.) and South American racers (Philodryas sp.)) are associated

with slowly or late evolving ecchymoses, systemic bleeding, coagulopathy and acute

kidney injury with minimal local envenoming. There have been fatalities from envenoming

by the African and Asian species, but South American non-front-fanged colubroid snakes

seem to be less dangerous. Mild local envenoming can be caused by bites of many

colubroid species, some of which are kept as pets in the West (for example, hognose

snakes Heterodon sp.) (Supplementary file S1)90,91.

[H3] Clinical time course. After bites by vipers (family Viperidae) and some cobras (family

Elapidae), local swelling is usually detectable within 2–4 hours and can extend rapidly to

reach its peak on the second or third day. Blistering appears within 2–12 hours and tissue

necrosis becomes obvious within 1 day of the bite. Sloughing of necrotic tissue and

17

secondary infections including osteomyelitis (infection of underlying bone) develop during

subsequent weeks or month. Complete resolution of swelling and restoration of normal

function in the bitten limb may take weeks. Systemic envenoming may be heralded by

vomiting or syncope within minutes of the bite. Coagulopathy and bleeding develop

within a few hours and can persist for ≥2 weeks in untreated patients. Neurotoxic signs

can progress to generalized flaccid paralysis and respiratory arrest within 30 minutes to a

few hours. Patients with neurotoxic envenoming usually recover within a few days with

assisted ventilation, but some may need respiratory support for as long as 10 weeks19,86,87.

[H2] Diagnosis

To deduce the nature and severity of envenoming, a sequential clinical history of

symptoms must be obtained and signs of envenoming rapidly elicited (Box 3), so that

appropriate, urgent, life-saving treatment can be given. Rapid clinical assessment must

include vital signs, measurement of postural blood pressure to exclude hypovolaemia,

formal testing for ptosis and signs of more advanced paralysis especially causing

respiratory failure, and examination for spontaneous systemic bleeding.

Patients usually know that they have been bitten, except those who suffer

painless nocturnal bites by kraits (Bungarus, Family Elapidae) while asleep92,93. Differential

diagnoses of snakebites include bites by arthropods (for example, spiders), lizards, rodents

or fish, stings (for example, by hymenopteran, scorpions or centipedes), or punctures by

plant spines or thorns, nails, splinters or other sharp objects. Definite snakebites that

result in negligible or no symptoms may have been ‘dry bites’ (transcutaneous bites

without envenoming94) inflicted by venomous snakes or bites by non-venomous species. If

the dead snake, or a photograph of it, is available, an expert herpetologist can identify the

species. Otherwise, descriptions by victims or bystanders can be helpful, supported by

recognition of evolving characteristic patterns (syndromes) of symptoms95 and aided by

biochemical measurements and imaging.

[H3] Laboratory and other investigations. Laboratory investigations can help to identify

systemic envenoming and aid the management of snake bites. Peripheral neutrophil

18

leucocytosis, indicating a general inflammatory response, confirms systemic envenoming.

Low haematocrit (volume percentage of red blood cells in blood) reflects severe

haemorrhage, whereas high haematocrit reflects haemoconcentration from leakage of

plasma into the tissues as a result of increased capillary permeability. Severe

thrombocytopenia is associated with severe bleeding diathesis and sometimes with

microangiopathic haemolysis, diagnosed by the presence of schistocytes in a blood film,

causing acute kidney injury. Incoagulable blood is a cardinal sign of systemic envenoming

by viperids, Australian and Oceanic elapids and non-front-fanged colubroid snakes. The

simple 20 minute whole blood clotting test (20WBCT) involves placing a few millilitres of

venous blood in a new, clean, dry, glass vessel, leaving it undisturbed at room

temperature for 20 minutes, and then tipping it once to see if it has clotted96,97. Lack of

clotting indicates severe consumption coagulopathy or an anticoagulant venom87.

Laboratory tests such as prothrombin and activated partial thromboplastin times, fibrin

degradation products and D-dimer are more sensitive indices of disseminated

intravascular coagulation and fibrinolysis. Levels of creatine kinase of >10,000 units per

litre indicate severe rhabdomyolysis. Blood urea or serum creatinine and potassium

concentrations should be measured in patients at risk of acute kidney injury. Urine should

be tested on admission for the presence of haemoglobin, myoglobin, blood and protein.

Electrocardiographic abnormalities include sinus bradycardia, ST–T changes, and

varying degrees of atrioventricular block or evidence of myocardial ischaemia. Myocardial

infarction can occur secondary to shock in patients with pre-existing coronary artery

disease. Echocardiography will detect pericardial effusion and myocardial dysfunction and

bleeding into the pleural and peritoneal cavities. Wound ultrasonography has also been

advocated to detect local bleeding98. CT and MRI is increasingly available for assessing

intracranial haemorrhages and infarcts.

[H3] Detection of venom. Detection and quantitation of venom antigens in body fluids of

snakebite victims, using enzyme immunoassays99–102 provides retrospective confirmation

of species diagnosis, predicts prognosis and is one measure of the effectiveness of

antivenom (also known as antivenin, antivenene, anti-snake-bite or anti-snake venom

19

serum) treatment. High concentrations of venom antigen (that is, from wound swabs or

wound aspirates) can be detected within 15–30 minutes, but commercial venom

detection kits are available only in Australia (produced by Seqirus, Parkville. Australia)103.

Venom detection kits are highly sensitive but insufficiently specific to distinguish between

venoms of closely related species. Detection of venom in a wound swab does not prove

that the patient has been envenomed and is not, on its own, an indication for antivenom

treatment. For retrospective species diagnosis, including forensic cases, tissue around the

fang punctures, wound and blister aspirate, serum and urine should be stored for enzyme

immunoassays. Detection of venom gland mRNA by reverse-transcription PCR104 or snake-

derived DNA in bite wound swabs105 are being developed as highly specific methods for

determining the identity of the biting species.

[H2] Prevention

The most effective method of preventing snakebites (Box 4) is through education

directed at high-risk communities and designed and driven from within those

communities86,87. A full range of media should be employed, including radio, TV, mobile

phone apps, social media, posters, puppet and drama performances and village-based

public meetings. Awareness of snakebite envenoming must be increased together with

advice on safer walking, working106 and sleeping107. Transport of bite victims to clinics

where they can receive medical care can be improved, even in areas that are inaccessible

to conventional ambulances, for example, using boats or volunteer village-based

motorcyclists108. Wasting time by visiting traditional therapists should be tactfully but

firmly discouraged.

[H1] Management

[H2] First-aid

Immediate first-aid after a bite should be done by the victim or bystanders.

Important elements are reassurance, immobilization of the whole body, especially the

20

bitten limb (to reduce dissemination of venom through veins and lymphatic system),

removal of rings and tight objects around the bitten limb and application of a pressure-

pad or pressure-bandage over the bite wound109,110. The patient must be transported

rapidly and preferably passively to the nearest place affording medical care. Pain should

be controlled using paracetamol (acetaminophen) or opioids, but not aspirin or NSAIDs

because of the risk of exaggerating bleeding problems. Minimizing the risk of fatal shock

and upper respiratory obstruction (through bulbar paralysis or fluid aspiration) during

transit is achieved by placing the victim in the recovery position and inserting an

oropharyngeal airway (a tube to maintain the airway). Ineffective and damaging

traditional treatments such as incisions, suction and tight tourniquets must be

discouraged. In suspected cases of neurotoxic envenoming by cobras, death adders

(Acanthophis sp.), some Latin American coral snakes (Micrurus sp.), and other elapids

whose venoms act mainly on postsynaptic receptors of the neuromuscular junction,

administration of atropine and neostigmine (an acetylcholinesterase inhibitor) to improve

neuromuscular transmission has been suggested as first-aid111.

[H2] Hospital management

Patients who claim to have been bitten by a snake should be admitted for a

minimum of 24 hours. They should be clinically assessed as described above. An

intravenous line and necessary resources for immediate resuscitation should be in place

before an in situ compression bandage or tourniquet is removed as this may precipitate

dramatic deterioration112. In patients who are breathless and centrally cyanosed (lips,

tongue and mucosae are blue because the blood is poorly oxygenated), the airway should

be restored and oxygen given by any possible means. If the patient is in shock, the foot of

the bed should be raised immediately, and intravenous fluid infused. Pain is variable but

might be very severe and should be treated appropriately, as described above.

Patients who initially present without evidence of envenoming can deteriorate

rapidly and unpredictably over minutes or hours. Published severity scores usually based

21

on arbitrary criteria are, therefore, inherently unreliable or even potentially dangerous.

However, several studies have shown that admission levels of venom antigenaemia

(concentration of venom antigens in serum or plasma detected by enzyme immunoassays)

were of prognostic value113,114. Snakebite victims should be carefully observed and their

blood pressure, pulse rate, level of consciousness, presence or absence of ptosis and

spontaneous bleeding, extent and magnitude of local swelling and urine output should be

measured. If clinical compartment syndrome (marked swelling of muscles contained in a

tight fascial compartment that might jeopardise blood supply) is suspected, intra-

compartmental pressure should be monitored. Assessing the level of consciousness of

patients with neurotoxic envenoming can be difficult because their generalized flaccid

paralysis makes the commonly used Glasgow Coma Scale misleading. For example, a

patient may not be able to open their eyes, speak or obey commands, but if

cardiorespiratory support is adequate and their paralysed upper eyelids are raised, they

may be found to be fully conscious and able to signal ‘yes’ or ‘no’ in response to simple

questions by flexing a finger or toe. After resuscitation and attempted species diagnosis,

the most critical management decision is whether the patient requires antivenom.

[H3] Antivenom. Antivenom is the only effective specific antidote for the systemic effects

of snakebite envenoming115. Antivenom consists of concentrated immunoglobulin of

horses, sheep or other large domesticated animals such as camels that have been

hyperimmunised with one or more venoms over periods of months to years. Worldwide,

most antivenom manufacturers refine the whole immunoglobulin G (IgG) extracted from

the animals’ plasma by enzyme digestion with pepsin to produce F(ab′)2 fragments, under

the assumption that removal of the Fc moiety from the antigen-binding (Fab) fragment

reduces the risk of adverse reactions. Others use papain to produce smaller Fab fragments

to improve safety and increase the speed of distribution throughout the body, but with

the disadvantage of rapid renal clearance of the antivenom so that recurrent envenoming

becomes a problem116. Some antivenoms consist of whole IgG molecules usually purified

by caprylic acid precipitation115. Venom-specific antibodies can be extracted by affinity

column purification, increasing safety but also cost.

22

Polyvalent (polyspecific) antivenoms are raised against the venoms of the most

medically important snake species in a particular geographical area. Examples are the

Indian antivenoms effective against the ‘big four’ national species, that is, Naja naja

(family Elapidae), Bungarus caeruleus (family Elapidae), Daboia russelii (family Viperidae)

and Echis carinatus (family Viperidae). By contrast, monovalent (monospecific)

antivenoms are raised against the venom of a single species, for example, European

ViperaTab effective for Vipera berus (family Viperidae).

Antivenoms have proved effective against many of the lethal and damaging effects

of venoms for more than a century. Antivenom administration can reverse anti-

haemostasis, hypotension and post-synaptic neurotoxicity and, if given early, prevent or

limit pre-synaptic neurotoxicity, rhabdomyolysis and local tissue necrosis19,86,87,115. In the

management of snakebites, the most important clinical decision is whether or not to give

antivenom. Antivenoms are highly specific and, therefore, will neutralize only the venoms

used in their production, together with those of a few related species, and so, in a

particular case of snakebite, an appropriate antivenom must be selected, based on

identification of the snake responsible for the envenoming. In addition, antivenoms are

costly, often scarce, poorly distributed in areas where they are most needed and may

require cold-chain for transport and storage117. Over the past few decades, several major

antivenom manufacturers (such as Syntex, Behringwerke and Sanofi-Pasteur) have

stopped production, mainly for commercial reasons, creating serious shortages in the

countries that they previously supplied, especially in Africa118. Only a minority of patients

bitten by a snake fulfil the criteria for antivenom use (Table 1).

Dosage is the same for adults and children. The initial dose would ideally be based

on clinical trial data119–123, but as these data are rarely available, the manufacturer’s

estimate of neutralising potency, based on rodent median effective dose, is usually the

guide. Dose is increased according to clinical estimates of severity of envenoming and is

repeated in the face of deteriorating neurotoxic or cardiovascular signs after 1–2 hours or

persistence of incoagulable blood after 6 hours. Administration is always intravenous,

over 10–60 minutes. Patients must be closely observed for early anaphylactic and

23

pyrogenic reactions especially during the first 2 hours after starting antivenom treatment.

The incidence and severity of these dose-related early adverse antivenom reactions can

be reduced by prophylactic adrenaline administration124. If reactions do occur, they should

be treated at the earliest sign (often itching and appearance of urticarial plaques,

restlessness, nausea, tachycardia or tachypnoea) with adrenaline by intramuscular

injection19,86,87,119–123.

[H3] Additional supportive treatment. Organ and system failures caused by envenoming

must be detected and treated (Table 2). Supraglottal or endotracheal intubation and

assisted ventilation either manually or by a ventilator is vital in cases of bulbar and

respiratory paralysis87. Especially in victims of species with predominantly postsynaptic

neurotoxins such as cobras (Naja; family Elapidae), death adders (Acanthophis; family

Elapidae) and some coral snakes (Micrurus; family Elapidae), acetylcholinesterase

inhibitors such as neostigmine, given with atropine, may improve neuro-muscular

transmission at least temporarily, but they are no substitute for antivenom111,125. Short-

acting acetylcholinesterase inhibitors (for example, edrophonium) or the ice pack test

(whereby an ice pack is applied to one upper eyelid in a patient with bilateral ptosis; the

ice lowers the local temperature and inhibits endogenous acetylcholinesterase) may be

used to predict response to endogenous cholinesterase inhibition126. Hypotension and

shock persisting after antivenom treatment is treated with cautious fluid volume repletion

and pressor drugs such as dopamine19,20. If acute kidney injury progresses despite

conservative management, renal replacement therapy is needed.

A tetanus toxoid booster should be given in all cases, but prophylactic antibiotics

are not justified127. However, if the wound has been incised or there are signs of tissue

necrosis, wound infection or local abscess formation, a broad spectrum antibiotic should

be given. Surgical debridement (removal of necrotic tissue) and skin grafting may be

needed in some cases, and some gangrenous digits or limbs might require amputation.

Painful, tender, tensely swollen, cold, cyanosed and apparently pulseless snake-bitten

limbs often appear to fulfill criteria for compartment syndrome (for example, anterior

tibial compartment), tempting surgeons to undertake fasciotomy (surgical procedure to

24

improve circulation by incising fascial compartments). Fasciotomy is rarely justified as

intracompartmental pressure usually remains within normal limits, and fasciotomy in

patients whose anti-haemostasis has not been corrected by adequate doses of antivenom,

has proved catastrophic. Unnecessary fasciotomy prolongs hospital stay and contributes

to long-term morbidity128.

Rehabilitation is a rare luxury for snakebite victims but is essential for helping to

restore function to the bitten limb, especially in children and agricultural workers, and to

ameliorate the chronic physical handicap that blights the lives of many snakebite

survivors.

[H2] Hospital discharge and follow-up

No snakebite victim should be discharged back to the same environment where

the bite occurred without receiving practical advice, preferably in the form of a printed

leaflet, about reducing the risk of further bites. At follow-up, patients should be checked

for late antivenom-related serum sickness 5–15 days after treatment, and chronic physical

and psychological sequelae of envenoming13,26. Persisting sequelae after snakebite are

common, and include tissue loss, amputations, contractures, arthrodesis (fusion fixation

of a joint), septic arthritis, hypertrophic and keloid scars, tendon damage, complications of

fasciotomies, chronic skin ulcers and osteomyelitis leading to malignant Marjolin

ulcers2,19,24,25,78,82,129. Visible and functional defects may lead to social stigmatization.

Acute kidney injury may lead to chronic renal failure and panhypopituitarism associated

with Russell’s viper (family Viperidae) envenoming to arrested puberty, amenorrhoea and

infertility. Persisting neurotoxic effects include mydriasis (pupil dilatation) and loss of

olfaction. Cerebral haemorrhages or thromboses may result in chronic neurological

deficits, and severe presynaptic neurotoxicity to increased risk of developing late

paralysis. Many survivors of snakebite complain of chronic or recurrent symptoms in the

bitten limb and attribute a wide variety of physical and mental problems to that

frightening and traumatic life event.

25

[H1] Quality of life

Snakebite envenomings occur mostly in impoverished settings11, affecting

underserved rural populations that, quite often, lack the appropriate resources to

confront this neglected disease. Snakebite alters the lives of victims, but also families and

communities because this disease largely affects people devoted to agricultural or

pastoralist activities whose economic performance, and that of their dependents, relies on

their good health. The death or incapacitation of someone responsible for the basic

economic support of his or her family can devastate the socioeconomic sustainability and

interpersonal stability of many people. Moreover, where medical treatment and especially

antivenom is not provided free of charge, the economic cost of snakebite can be

catastrophic — creating debt, forcing asset liquidation, and driving families further into

the cycle of poverty130,131.

Although many snake bites are fatal, far more leave behind victims who experience

horrifically debilitating disfigurement and long-term disability. Without timely access to

health systems with adequate resources and capacity the consequences of a snakebite

envenoming may include various types of sequelae2,7,129,132,133. Where health systems

falter, affected people as well as their families and communities must deal with these

consequences on their own often with severe consequences, often expanding the toll of a

single snakebite envenoming.

This disease also exerts a heavy psychological impact, a phenomenon that has not

received attention in medical research until recently26,134. The lack of effective diagnosis

and treatment, and poor follow-up of affected people, even when they are treated in

health facilities, often results in psychological consequences that remain untreated, thus

affecting patients and their families in ways that go beyond the biomedical aspects of the

disease26,134.

26

Rolling back the impact of snakebite envenoming on quality of life requires

concerted and coordinated efforts spanning entire health systems. Snakebite envenoming

is a ‘tool-ready’ disease, in the sense that effective therapy (that is, antivenom) exists, and

other cross-cutting interventions are available to strengthen health systems, empower

communities and encourage policy change. Implementing an effective action plan to

control snakebite envenoming will lead to a situation where snakebite victims are treated

rapidly with safe, effective antivenoms by trained health staff, followed up with ancillary

treatment, together with psychological, social and economic support after hospital

discharge. Public and private organizations, as well as community-based groups, should

follow up and help people affected by sequelae of snakebites.

[H1] Outlook

[H2] Diagnosis

Deciding on when to start antivenom treatment can be difficult, particularly for

inexperienced physicians, as signs of envenoming and their time of onset vary by snake

genus, the amount of venom injected, the site of injection and the age and health of the

victim. In addition, current clinical guidelines recommend withholding antivenom

administration until symptoms of systemic envenoming are detected in snakebite victims.

Rapid, affordable, point-of-care (bedside) diagnostic kits providing physicians in rural

hospitals with information enabling earlier detection of envenoming and identification of

the biting species are urgently needed, to enable earlier treatment with antivenom and

anticipate likely clinical course and need for supportive therapy (for example, ventilation).

An experimental lateral flow assay has been developed to identify envenoming by

two Indian snakes; the assay uses antivenom to detect circulating venom proteins135. The

near-ubiquitous delivery of PLA2s into the circulation of snakebite victims has been

suggested as a marker to detect systemic envenoming136. A PCR-based approach has also

been reported as a possible diagnostic tool for detecting venom DNA in and around the

27

bite site, but may be less useful for detecting circulating venom as a marker of systemic

envenoming105.

[H2] Management of systemic effects

Antivenom has been the primary treatment of systemic snake envenoming for over

a century. Whilst life-saving, antivenoms still have therapeutic limitations137,138.

Conventional antivenom has limited efficacy against some effects of envenoming, such as

local tissue damage, and, when treatment is delayed, presynaptic neurotoxicity. In

addition, only 10–15% of IgG in a vial of antivenom is specific to venom proteins, because

the animals used for antivenom manufacture already have mature immune systems, and

hyper-immunisation with venom is unable to engender higher venom-specific IgG titres.

Finally, the greater the phylogenetic disparity between the snakes whose venoms are used

for immunisation, the greater the number of IgG specificities generated in the venom-

immunised animals. This means that the proportion of total IgG targeting the venom of

any one snake is small, and consequently more vials are needed to achieve clinical cure.

This creates a therapeutic paradox because each extra vial of antivenom increases both

the risk of potentially severe antivenom-induced adverse effects and treatment cost. Thus,

highly efficient, polyspecific antivenoms are needed, without compromising safety and

affordability.

The rapidly increasing availability of snake venom proteomes and venom gland

transcriptomes40 have provided essential information on venom protein composition and

inferred function of the proteins. Together with DNA sequence information, this allows

guided development of IgGs that target only the most toxic venom proteins. One

approach is to analyse transcriptome data of each toxin group expressed in snake venom

(of several snake families) to identify antigenic sequence motifs (epitopes) with the

greatest cross-species and cross-genera sequence conservation. These motifs are

manipulated to construct a synthetic epitope string designed to generate multiple distinct

toxin group-specific IgGs capable of neutralizing that the entire toxin group, irrespective of

the snake species139–141. The intent is to pool all the toxin group-specific IgGs to create a

28

polyspecific antivenom. To address the dose–efficacy challenge, the current intent is to

isolate B cells from epitope string-immunized mice, and extract, screen and manipulate

the IgG-encoding genes to prepare monoclonal antibodies to produce an antivenom

consisting only of IgG capable of polyspecifically neutralising the toxins present in the

venoms of a defined group of snakes. Humanizing these monoclonal antibodies also offers

substantial safety advantages over the current antivenoms142.

Another toxin-specific recombinant antivenom approach is based on screening

bespoke high-density toxin-specific microarrays to identify the most medically-important

venom toxin epitopes bound by clinically-effective antivenoms143. This toxin-focused

approach aligned with the production of human or humanized IgGs (or fractions thereof,

for example, single-chain variable fragments) using new biotechnology production

processes may yield antivenoms at a cost similar to conventional antivenoms in the

future144, but with a much-improved dose-efficacy. International collaborative efforts are

underway in these research and development topics.

Chemical inhibitor research is also being promoted142 as an overlooked but

potentially source of drugs to treat snakebites. For example, a recent drug repurposing

study identified a potent, broad-spectrum inhibitor of the nearly-ubiquitous group of

venom PLA2s145. Other efforts have been directed at the synthesis of nanoparticles which

could sequester and neutralize venom toxins146. If successful, the chemical outputs of

these approaches will likely have substantial cost and logistic advantages.

A pharmacological study reported that an ointment containing a nitric oxide donor,

which impedes the intrinsic lymphatic pump, delays ingress of venom proteins into the

systemic circulation and improved survival of venom-injected rats147. This first aid-focused

application is likely to be effective in predominantly neurotoxic venoms, but needs to be

excluded for envenoming by those snakes that cause local tissue destruction.

[H2] Management of local effects

29

No medicinal treatment of the local tissue-destructive effects of envenoming

exists. Antivenom, unless it is administered very soon after the bite, is largely ineffective in

preventing the rapidly manifested destruction of tissues by venom. Chemical inhibitors

have potential as a treatment for venom-induced local tissue destruction owing to their

low-cost, thermostability and rapid tissue-distribution dynamics. Small peptidomimetic

matrix metalloproteinase inhibitors have shown exciting, repurposed, ability to neutralize

SVMP-induced haemorrhage and dermonecrosis148. It is not inconceivable that combining

inhibitors of SVMPs and PLA2s145,146,148,149 may produce an affordable, rapid, polyspecific,

and effective treatment of venom-induced tissue damage.

Other groups are pursuing a recombinant approach using single-domain VHH

antibody fragments, based upon the demonstration that an experimental VHH antivenom

(prepared from the heavy chain-only IgG3 fraction of a venom-immunised dromedary

camel) proved the most dose-effective antivenom137 to neutralize various toxic effects of

the West African saw-scaled viper (Echis ocellatus). The small size, thermostability and

toxin-specificity of camelid VHH promote them as exciting therapeutic candidates for

preventing the tissue necrotic effects of envenoming for saw-scaled vipers and other

snake venoms150,151.

[H2] Treatment availability and accessibility

There is a current crisis in antivenom provision in various regions, particularly sub-

Saharan Africa and parts of Asia10,138,152. The market failure of two of the most effective

polyspecific antivenoms manufactured for Africa in the past118,138,153 underscores the fact

that the commercial constraints associated with marketing non-subsidised conventional

antivenoms of high cost but limited demand can result in important, life-threatening

therapeutic gaps. It also reinforces the need to incorporate commercial realities into the

design of innovative approaches to generate antivenoms with improved polyspecific

efficacy, safety and affordability137,138. The research and development tasks discussed

above need to be complemented by research and innovation in the public health realm

that aim to improve the availability and accessibility of antivenoms, which demands

research in subjects such as health economics, provision of health services, and other

30

social science-related topics. The long-term goal is to develop knowledge-based policies

ensuring that people suffering snakebite envenomings receive safe and effective

antivenoms in a timely fashion. New treatments should be affordable and available to

invariably remote, impoverished snakebite victims138,152. In addition, innovative treatment

and diagnostic research needs to be complemented with equally important research on

ways to ensure affected communities can better access effective healthcare, and indeed

upon affordable and appropriate means by which they can reduce their likelihood of being

bitten.

[H2] Venoms as a source of therapy

Some of the targets of toxins are also dysregulated in a number of human diseases,

such as thrombotic disorders, vascular pathologies, neurodegenerative diseases and

inflammatory conditions. The study of venom toxins is of growing interest for the

pharmacological and biotechnological communities, as venoms are increasingly

recognized as a rich source for lead compounds that can drive the development of

biotechnological tools and novel biotherapeutics154. Determining the molecular landscape

of snake venoms represents the necessary first step towards these basic and applied

goals.

[H2] Global efforts to reduce impact

For decades, a major barrier to effectively reducing the worldwide impact of

snakebite envenoming has been the lack of inclusion of this disease on the global public

health agenda. The absence of appropriate prioritization and resourcing of control efforts

has seen snakebite envenoming fail to receive proper attention by international and

national health authorities, fall off research priority lists and public health agendas, and

lose the interest of the pharmaceutical industry. However, the decision of the WHO in

June 2017 to include snakebite envenoming in the category A of its list of Neglected

Tropical Diseases is a significant step forward in the global struggle to reduce the impact

of this pathology. This is an essential advance that is necessary to raise the profile of

snakebite envenoming in the eyes of Member State governments, donors and other

31

stakeholders, and to empower WHO to provide the leadership needed to coordinate a

global control programme. WHO is the only organization with the political, operational

and policy reach to drive an integrated global strategy involving multiple actors and a

multidisciplinary approach, and this is clearly demonstrated by the success in combating

other neglected tropical diseases in the five years since the London Declaration on

neglected tropical diseases was enacted to support the WHO’s 2020 Roadmap.

Inclusion of snakebite envenoming in the neglected tropical disease category will

focus attention on concentrated efforts to control snakebite through an integrated and

intensified disease management strategy incorporating several key elements, ranging

from prevention, to improved primary treatment, rehabilitation and investment in

research that can unlock new diagnostic and therapeutic pathways, or enhance

surveillance and resource deployment (Box 4).

The tasks included in Box 4 are by no means an exhaustive list of all the potential

interventions available to immediately bring the burden of snakebite envenoming under

effective control, but it exemplifies the key approaches that will drive the process.

Broadening the current focus which revolves around improving treatment with

antivenoms, measuring burden and assessing the consequences to take in a more holistic

strategy that begins with community engagement, empowerment and education and

spans all stages of treatment, reporting, therapeutic and diagnostic translational research,

policy, training and research is essential.

This integrated plan for controlling snakebite envenoming requires international

cooperation, through a multitiered roadmap coordinated by WHO, involving many

stakeholders: the scientific and technological research community; antivenom

manufacturers and developers; health regulatory agencies; public health authorities at

national, regional and global levels, and health professional organizations; international

health foundations and advocacy groups, such as the Global Snakebite Initiative10, Health

Action International, and others; and organizations of the civil society in countries of high

incidence of snakebites. Realizing such as comprehensive strategy requires considerable

32

resources, and it is incumbent on United Nation Member States, donor organizations,

snakebite experts and other actors to wholeheartedly support the leadership of WHO as it

moves forward to implement effective control programmes and projects.

33

Highlighted references

Mohapatra, B. et al. Snakebite mortality in India: a nationally representative mortality

survey. PLoS Negl. Trop. Dis. 5, e1018 (2011).

A nationally representative study of snakebite mortality in India demonstrating that the

magnitude of the problem in terms of mortality is much higher than previously

thought.

Williams, D. et al. The Global Snakebite Initiative: an antidote for snake bite. Lancet 375,

89-91(2010).

This paper describes the launching of the first organization to confront snakebite

envenoming from a global perspective.

Harrison, R.A. et al. Snake envenoming: a disease of poverty. PLoS Negl. Trop. Dis. 3, e569

(2009).

The relationship between snakebite envenoming and poverty is highlighted in this

study.

Habib, A.G. et al. Snakebite is under appreciated: appraisal of burden from West Africa.

PLoS Negl. Trop. Dis. 9, e0004088 (2015).

This study analyzed the impact of snakebite envenoming in terms of disability-adjusted

life years in 16 countries in sub-Saharan Africa.

Calvete, J.J. Venomics: integrative venom proteomics and beyond. Biochem. J. 474, 611-

634 (2017).

A comprehensive view of the complexity of snake venoms and its biological and medical

implications.

Montecucco C., Gutiérrez, J.M. & Lomonte, B. Cellular pathology induced by snake venom

phospholipase A2 myotoxins and neurotoxins: common aspects of their

mechanisms of action. Cell. Mol. Life Sci. 65, 2897-2912 (2008).

A review of the mechanisms by which venom phospholipases A2 induce myotoxicity and

neurotoxicity.

34

Escalante, T., Rucavado, A., Fox, J.W. & Gutiérrez, J.M. Key events in microvascular

damage induced by snake venom hemorrhagic metalloproteinases. J. Proteomics

74, 1781-1794 (2011).

This review summarizes the mechanisms by which zinc-dependent venom

metalloproteinases induce microvascular damage and hemorrhage.

World Health Organization Regional Office for Africa. Guidelines for the Prevention and

Clinical Management of Snakebite in Africa (WHO, Brazzaville, 2010)

http://www.afro.who.int/pt/grupos-organicos-e-programas/2731-guidelines-for-

the-prevention-and-clinical-management-of-snakebite-in-africa.html

WHO Guidelines for snakebite envenoming in Africa, to be used in the training of health

staff on the correct diagnosis and management of envenomings.

Sharma, S.K. et al. Effectiveness of rapid transport of victims and community health

education on snake bite fatalities in rural Nepal. Am. J. Trop. Med. Hyg. 89, 145-

150 (2013).

A study describing a successful intervention at the community level aimed at improving

the access of envenomed patients to health facilities.

World Health Organization. WHO Guidelines for the Production, Control and Regulation of

Snake Antivenom Immunoglobulins. WHO, Geneva, 2010.

http://www.who.int/bloodproducts/snake_antivenoms/en/A detailed account on the

main aspects related to the production and control of antivenoms, containing

valuable information for manufacturers, researchers, and regulatory agencies.

Abubakar, I.S. et al. Randomised controlled double-blind non-inferiority trial of two

antivenoms for saw-scaled or carpet viper (Echis ocellatus) envenoming in Nigeria.

PLoS Negl. Trop. Dis. 4, e767 (2010).

Código de campo cambiado

35

An example of a clinical trial based on a randomized controlled double-blind design for

the evaluation of efficacy and safety of antivenoms in the treatment of

snakebite envenomings.

Vaiyapuri, S. et al. Snakebite and its socioeconomic impact on the rural population of

Tamil Nadu, India. PLoS ONE 8, e80090 (2013).

A description of socioeconomic consequences of snakebites in an impoverished rural

setting.

Harrison, R.A., Cook, D.A., Renjifo, C., Casewell, N.R., Currier, R.B. & Wagstaff, S.C.

Research strategies to improve snakebite treatment: challenges and progress. J.

Proteomics 74, 1768-1780 (2011).

This review discusses some of the main research areas that need to be developed in

order to generate novel diagnostic and therapeutic tools to confront snakebite

envenoming.

Laustsen, A.H. et al. From fangs to pharmacology: The future of snakebite envenoming

therapy. Curr. Pharm. Des. 22, 5270-5293 (2016).

A summary of novel therapeutic alternatives to approach snakebite envenoming,

including recombinant antibodies and natural and synthetic venom inhibitors.

Acknowledgments

The authors thank Dr. Jeronimo Bravo (Instituto de Biomedicina de Valencia, CSIC) for

assisting in the elaboration of the structural models shown in Figure 3.

Author contributions

Introduction (J.M.G.); Epidemiology (A.G.H.); Mechanisms/pathophysiology (J.J.C. and

J.M.G.); Diagnosis, screening and prevention (D.A.W.); Management (D.A.W.); Quality of

life (D.J.W.); Outlook (J.M.G., R.A.H. and D.J.W.); Overview of the Primer (J.M.G.)

Código de campo cambiado

Código de campo cambiado

36

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and

institutional affiliations.

How to cite this Primer

Gutiérrez J. M. et al. Snakebite envenoming. Nat. Rev. Dis. Primers 3, XXXX (2017).

37

Box 1 | Technologies to analyse snake venom composition.

First-generation venom proteomics (venomics) relied on reversed-phase high-

performance liquid chromatography separation and quantification of venom components

followed by in-gel digestion of the protein bands corresponding to the chromatographic

peaks. Protein bands were resolved by sodium dodecyl sulfate polyacrylamide gel

electrophoresis, de novo tandem mass spectrometry sequencing of the peptides (usually

produced by trypsin digestion) and database searching by Basic Local Alignment Search

Tool (BLAST) analysis155. An experienced researcher typically completes this bottom-up

analysis of a venom composed of 35 chromatographic fractions and 60-70 electrophoretic

bands in 6-8 weeks. The bottleneck of this technique is the quality of the tandem mass

spectrometry fragmentation spectra and the presence in the databases of a sequence

homologous to the de novo derived amino acid sequence tag for the digested peptide

ions. Peptide-centric approaches provide incomplete sequence coverage, and in the

absence of a species-specific venom gland transcriptome, only provide information on

which known protein families are present in the venom without distinguishing between

different proteoforms (different molecular forms of a protein) or toxin isoforms (proteins

encoded by closely related genes).

Top-down mass spectrometry using high-resolution ion-trapping mass spectrometry in

conjunction with comprehensible species-specific venom gland database matching156,157

has the potential to overcome this shortcoming158,159. The top-down mass spectrometry

configuration enables label-free relative quantification of the different protein species in

line with their identification. However, mass spectrometry is not inherently quantitative

because of differences in the ionization efficiency and/or detectability of the different

components in a given sample. This analytical limitation has sparked the development of

methods to determine absolute abundance of proteins in samples. Inductive coupled

plasma mass spectrometry with online 34S isotope dilution analysis for the absolute

quantitative analysis has been recently successfully applied to quantify the toxins of the

Mozambique spitting cobra, Naja mossambica160.

38

Box 2. Toxic activities of main protein families in snake venom

- Phospholipases A2: local and systemic myotoxicity, pain, damage to lymphatic vessels,

oedema, neurotoxicity, nephrotoxicity and haemolysis

- Snake venom metalloproteinases: haemorrhage, myonecrosis, extracellular matrix

degradation, blistering, pain, oedema and cardiovascular shock, nephrotoxicity and

coagulopathy

- Hyaluronidases: extracellular matrix degradation

- Three-finger toxins: cytotoxicity, necrosis and neurotoxicity

- Dendrotoxins: neurotoxicity

- Snake venom serine proteinases: coagulopathy, oedema and hypotension

- Vasoactive peptides (for example, bradykinin-potentiating peptides): hypotension

- Disintegrins: inhibition of platelet aggregation

- C-type lectin-like proteins: inhibition or promotion of platelet aggregation and

thrombocytopenia

- Cysteine-rich secretory proteins: smooth muscle paralysis

- Small basic myotoxic peptides: muscle contracture

- Natriuretic peptides: hypotension

- Sarafotoxins: cardiotoxicity

39

Box 3 | Initial clinical history from a snakebite victim.

The following five brief questions have proved helpful for rapid assessment of snakebite

victims.

Where were you bitten? Examine puncture marks, swelling, inflammation,

bruising, persistent bleeding and evidence of pre-hospital traditional treatment at

the site of the bite.

When were you bitten? Note that if the bite or sting was very recent, there may

not have been time for signs of envenoming to develop.

What were you doing when you were bitten? Note that the circumstances of the

bite might be diagnostic.

Where is the snake that bit you or what did it look like? If it was killed but was left

behind, send someone to bring it, in whatever condition. Ask if a photo of the

snake was taken.

How are you feeling now? Check whether any symptoms of envenoming have

developed.

40

Box 4 | Recommendations for an integrated global strategy to confront snakebite

envenoming8,138

Actively prevent contact and implement educational programmes that detail safe

methods for carrying out chores or work that pose a high risk of encountering snakes.

For example, when collecting firewood, picking up piles of raked leaves, harvesting

crops or hunting, appropriate footwear should be worn and a torch or flashlight

should be used to illuminate the path at night. Risk during sleeping can be minimized

by snake-proofing dwellings and using protective (mosquito) bed-nets or raised

sleeping platforms.

Establish effective, safe and affordable first aid that delays the evolution of clinical

illness and improve transport such that victims of envenoming reach health care

facilities for diagnosis and treatment as quickly as possible.

Train health professionals to implement standard treatment protocols and

algorithms, and provide diagnostic tools to strengthen health systems and improve

medical management of snakebite victims.

Develop a robust system for hospital and community-level surveillance of snakebite

envenoming, including mandatory reporting of snake-bite victims seeking treatment,

notifications of deaths and active detection of cases through community surveys and

outreach programmes.

Establish standard definitions and measurements to facilitate the accurate collection

and comparability of data.

Improve access and distribution of medicines, especially of safe, effective and

affordable antivenoms by implementing programmes that reduce prices through

collective bulk purchasing by consortia (governments, non-governmental

organizations and aid donors).

Collaborate with the WHO Essential Medicines and Health Products department to

improve manufacturing and quality control systems of antivenoms, which will enable

optimization of the production pipeline.

41

Strengthen health systems to improve the treatment of snakebite envenoming in

health facilities from admission through to discharge and follow-up. Follow-up is

especially important in patients with local tissue injury and disability.

Invest in the improvement of existing antivenom treatments and the development of

innovative future treatments.

Collaborate with the WHO Neglected Tropical Diseases department to implement a

wide range of interventions in the control, prevention and treatment of snakebite

envenoming.

42

Figure 1. Venomous snakes. A | Schematic illustration of the venom system in a snake of

the family Viperidae (viperids). Venom is synthesized and stored in a specialized gland.

When a bite occurs, venom is expelled through the action of a compressor muscle that

surrounds the venom gland, and is delivered by a duct to the fangs through which it is

injected in the tissues of the victim. Snake species responsible for a highest mortality

owing to snakebite envenoming are Echis ocellatus (carpet viper; family Viperidae; part b)

and Bitis arietans (puff adder; family Viperidae; part c) in Africa, Naja naja (cobra; family

Elapidae; part d), Bungarus caeruleus (common krait; family Elapidae; part e), Daboia

russelii (Western Russell’s viper; family Viperidae; part f) in Asia, Bothrops atrox (common

lancehead; family Viperidae; part g) and Bothrops asper (terciopelo; family Viperidae; part

h) in the Americas and Oxyuranus scutellatus (Papuan taipan; family Elapidae; part i) in

Oceania. Many other snake species are also capable of inducing life-threatening

envenomings. Photos b–h by David A. Warrell, and photo i by David J. Williams (used with

permission). [Note to copy editor: we have iLTPs for these images from David Warrell; please

insert the appropriate credit line as per style.]

Figure 2. Geographical distribution of the estimated number of snakebite envenomings

and deaths. Data shown constitute a rough approximation of the estimated range of

envenomings and deaths given that, in many countries, reliable information on snakebite

morbidity and mortality are lacking, resulting in underreported data of this neglected

tropical condition. The highest impact of snakebite envenomings occurs in Asia, sub-

Saharan Africa, Latin America and parts of Oceania. Based on estimates from5,12.

Figure 3. Toxin levels in venom of Viperidae and Elapidae. The graph highlights the

protein levels (% of the total venom proteome) and the distinct distribution of the most

abundant toxin families in venoms of snake species from the families Viperidae

(subfamilies Viperinae and Crotalinae) and Elapidae (subfamilies Elapinae and

Hydrophiinae). Bars are colour-coded according to the most relevant biological activities

of the corresponding toxin family36,40. Colour gradients indicate concentration

dependency of the biological effect (same colour) or different effects (multiple colours;

43

that is, some toxins may exert one effect at low doses and another effect at high doses).

The crystallographic or NMR structures of some members of each protein family are also

shown. SVMP, snake venom metalloproteinases (Protein Data Bank (PDB) accession 3DSL

for class PIII and PDB accession 1ND1 for class PI are shown); PLA2, phospholipases A2

(PDB accession 1TGM for the monomer and PDB accession 3R0L for the dimer); SVSP,

snake venom serine proteinases (PDB accession 1OP0); 3FTx, three-finger toxins (PDB

accession 1IJC); DTx, dendrotoxins (PDB accession 1DTX); CTL, C-type lectin-like proteins

(PDB accession 1IXX); CRISP, cysteine-rich secretory proteins (PDB accession 3MZ8); LAO,

L-amino acid oxidases (PDB accession 2IID); Myo, low molecular mass myotoxins (PDB

accession 4GV5). More information on the crystal structures shown and their source can

be found in Supplementary file 2.

Figure 4. Immunoaffinity capturing antivenomics protocol. Whole venom is applied to an

immunoaffinity column packed with antivenom antibodies that are immobilized onto

Sepharose beads. After eluting the non-retained venom components, the

immunocaptured proteins are eluted. Comparison of the reversed-phase high-

performance liquid chromatographs of the components of whole venom, the fraction

recovered from the affinity antivenom column (immunoretained fraction) and the non-

immunocaptured venom fraction (non-immunoretained fraction) can provide qualitative

and quantitative information on the set of toxins bearing antivenom-recognized epitopes

and those toxins exhibiting poor immunoreactivity.

Figure 5. Action of snake venom toxins on different body systems. Venoms exert a wide

spectrum of toxic activities in the body, and the predominant deleterious actions depend

on venom composition. Elapid venoms, and some viperid venoms, induce neuromuscular

paralysis. Most viperid venoms, and some elapid venoms, inflict prominent local tissue

damage. Viperidae venoms cause systemic haemorrhage, which, together with increased

vascular permeability, can lead to cardiovascular shock. Viperid and some elapid and

colubroid venoms act at various levels of the coagulation cascade and on platelets,

thereby affecting haemostasis. Some venoms cause generalized muscle breakdown (that

44

is, rhabdomyolysis). Acute kidney injury often develops in envenomings, owing to a

multifactorial pathogenesis.

Figure 6. Clinical effects of snake venoms. A | Swelling and blistering following a bite on the

dorsum of the foot by a jararaca (Bothrops jararaca, family Viperidae) in Brazil. B | Swelling,

blistering and gangrene of the hand, which required amputation, and extensive ecchymoses

(discoloration of the skin owing to bleeding under the skin) following a bite of the Malayan pit-

viper (Calloselasma rhodostoma, family Viperidae) in Thailand. C | Bleeding gums, a cardinal sign

of failure of haemostasis, in a patient bitten by a saw-scaled viper (Echis ocellatus, family

Viperidae) in Nigeria. D | Bilateral conjunctival oedema (chemosis), indicating a generalized

increase in capillary permeability, following a bite of Eastern Russell’s viper (Daboia siamensis,

family Viperidae) in Myanmar. E | Bilateral ptosis (paralysis of both upper eyelids) and F | external

ophthalmoplegia (paralysis of the eye muscles; the patient cannot look to the right), in patients

bitten by Papuan taipans (Oxyuranus scutellatus, family Elapidae) in Papua New Guinea. G |

Extensive necrosis of skin and sub-cutaneous tissue following a bite on the elbow by a black-

necked spitting cobra (Naja nigricollis, family Elapidae) in Nigeria. All images courtsey David A.

Warrell. [Note to copy editor: we have iLTPs for these images from David Warrell; please insert

the appropriate credit line as per style.]

45

Table 1. Criteria for antivenom treatment

Clinical criterion Clinical evidence Additional treatment

Shock with or without hypovolaemia

Low or falling blood pressure (with

postural drop)

Increasing pulse rate

Prostrated or collapsed, cold, pale or

peripherally cyanosed

If clinically hypovolaemic, administer

volume repletion and/or pressor drugs*

Spontaneous systemic bleeding (at sites

distant from the bite)

Bleeding of the gums, nose,

gastrointestinal tract and/or

urogenital tract

Stroke

If massive or threatened (for example, in

case of imminent surgery, child birth),

administer fresh frozen plasma and/or

other blood products*

Incoagulable blood

Persistent bleeding from trauma sites

Positive 20WBCT

Altered laboratory blood coagulation

profile

If massive or threatened (for example,

pre-delivery in pregnant women or before

surgery), administer fresh frozen plasma

and/or other blood products*

Neurotoxicity

Bilateral ptosis

External ophthalmoplegia

Descending paralysis

Restore and secure airway (consider

endotracheal intubation and assisted

ventilation)

Trial of anticholinesterase or ‘ice-pack

test’‡

Black urine

Macroscopic or microscopic evidence

of haemoglobin or myoglobin in the

urine (urine reagent strip testing)

Exclude haematuria

Restore fluid homeostasis

Alkalinize urine

High risk of actute kidney injury Oliguria/anuria

Rising plasma creatinine and urea

(especially after bites by high risk

species)

Fluid challenge, conservative treatment or

renal replacement

Extensive local swelling or high risk of

tissue necrosis

Rapidly-progressive local swelling

(especially bites on digits)

Monitor intra-compartment pressure

Administer antibiotics*

Treatment with specific antivenom is indicated if one or more of the following criteria are fulfilled. *See Table 2. ‡ Application of an ice

pack to one upper eyelid in a patient with bilateral ptosis; the ice lowers the local temperature and inhibits endogenous

acetylcholinesterase; if positive, anticholinesterase can be administered. 20WBCT, 20 minute whole blood clotting test.

46

Table 2 | Approved drugs for supportive care of snakebite victims in addition to antivenom

Drug or treatment Indication Comment

Adrenaline Prevention and treatment of early anaphylactic antivenom reactions

Treatment of early autopharmacological anaphylactic reactions due to envenoming or acquired venom hypersensitivity

Prophylactic: subcutaneous treatment with a low dose before antivenom treatment.

Therapeutic: intramuscular injection.

Analgesics (for example, paracetamol or opioids)

Routine analgesia Pain is an underestimated symptom; most snake bites are painful, some are agonizing.

Aspirin or NSAIDs should not be given because of bleeding risks.

Antibiotics Bite wounds that are necrotic or that have been tampered with

Clinically-obvious wound infection (for example, abscess), distinguished from inflammatory effects of envenoming

Prophylactic antibiotics are not indicated unless the wound is necrotic or has been tampered with.

The choice of antibiotic is guided by bacterial culture result or, if wound is necrotic, immediate broad spectrum cover to include Clostridium sp. and other anaerobic bacteria.

Acethylcholinesterases inhibitors (for example, neostigmine) after atropine to block muscarinic effects

To prolong biological half-life of acethylcholine at peripheral neuro-muscular junctions.

For neurotoxic envenoming, especially by species with post-synaptic toxins

Administered after positive result of test dose of short-acting edrophonium (a reversible acetylcholinesterase inhibitor) or positive ice-pack test*

Antihistamine H1 blocker (for example, chlorphenamine)

Early anaphylactic reactions (after adrenaline) to antivenom (intravenous administration)

Mild late serum-sickness-type antivenom reactions (oral or parenteral administration)

Ineffective for prophylaxis or for severe anaphylaxis

Blood products (for example, fresh frozen plasma or cryoprecipitates)

Accelerate restoration of haemostasis in case of imminent surgery, child birth or severe bleeding

Conservative treatment of antihaemostatic disorders when no specific antivenom is available

Unless venom procoagulants are neutralized with specific antivenom, administered clotting factors carry the risk of promoting thrombus-formation with potentially-fatal consequences

Corticosteroids Severe late serum-sickness (oral prednislolne)

Suspected or confirmed acute pituitary or adrenal failure (intravenous hydrocortisone)

Should not be used routinely for snake bites and have no role in treating early anaphylactic antivenom reactions.

Do not reduce the risk of recurrent anaphylaxis

Pressor drugs agents (for example, noradrenaline, vasopressin and dopamine)

Low or falling blood pressure despite fluid volume replacement and specific antivenom administration

Severe anaphylaxis refractory to adrenaline and fluid volume repletion

Preferable to excessive fluid replacement, which may precipitate volume-overload pulmonary oedema

Tetanus toxoid To boost immunity against tetanus toxins in all cases

Also reassuring for non-envenomed patients.

Use anti-tetanus serum for neglected necrotic wounds in un-immunised patients

*Application of an ice pack to one upper eyelid in a patient with bilateral ptosis; the ice lowers the local temperature and inhibits endogenous acetylcholinesterase; if positive, anticholinesterase can be administered.

1. Calvete, J. J. Proteomic tools against the neglected pathology of snake bite envenoming.

Expert Rev. Proteomics 8, 739–758 (2011).

2. Warrell, D. A. Snake bite. Lancet 375, 77–88 (2010).

47

3. Pyron, R., Burbrink, F. T. & Wiens, J. J. A phylogeny and revised classification of

Squamata, including 4161 species of lizards and snakes. BMC Evol. Biol. 13, 93 (2013).

4. Hsiang, A. Y. et al. The origin of snakes: revealing the ecology, behavior, and evolutionary

history of early snakes using genomics, phenomics, and the fossil record. BMC Evol. Biol.

15, (2015).

5. Chippaux, J. P. Snake-bites: appraisal of the global situation. Bull. World Health Organ. 76,

515–24 (1998).

6. Mohapatra, B. et al. Snakebite Mortality in India: A Nationally Representative Mortality

Survey. PLoS Negl. Trop. Dis. 5, e1018 (2011).

7. Chippaux, J.-P. Estimate of the burden of snakebites in sub-Saharan Africa: A meta-analytic

approach. Toxicon 57, 586–599 (2011).

8. Gutiérrez, J. M., Williams, D., Fan, H. W. & Warrell, D. A. Snakebite envenoming from a

global perspective: Towards an integrated approach. Toxicon 56, 1223–1235 (2010).

9. Gutiérrez, J. M., Theakston, R. D. G. & Warrell, D. A. Confronting the Neglected Problem

of Snake Bite Envenoming: The Need for a Global Partnership. PLoS Med. 3, e150 (2006).

10. Williams, D. et al. The Global Snake Bite Initiative: an antidote for snake bite. Lancet 375,

89–91 (2010).

11. Harrison, R. A., Hargreaves, A., Wagstaff, S. C., Faragher, B. & Lalloo, D. G. Snake

Envenoming: A Disease of Poverty. PLoS Negl. Trop. Dis. 3, e569 (2009).

12. Kasturiratne, A. et al. The Global Burden of Snakebite: A Literature Analysis and Modelling

Based on Regional Estimates of Envenoming and Deaths. PLoS Med. 5, e218 (2008).

13. Habib, A. G. et al. Snakebite is Under Appreciated: Appraisal of Burden from West Africa.

48

PLoS Negl. Trop. Dis. 9, e0004088 (2015).

14. Alirol, E., Sharma, S. K., Bawaskar, H. S., Kuch, U. & Chappuis, F. Snake Bite in South

Asia: A Review. PLoS Negl. Trop. Dis. 4, e603 (2010).

15. Sankar, J., Nabeel, R., Sankar, M. J., Priyambada, L. & Mahadevan, S. Factors affecting

outcome in children with snake envenomation: a prospective observational study. Arch. Dis.

Child. 98, 596–601 (2013).

16. Stahel, E. Epidemiological aspects of snake bites on a Liberian rubber plantation. Acta Trop.

37, 367–74 (1980).

17. Warrell, D. A. et al. Randomized comparative trial of three monospecific antivenoms for

bites by the Malayan pit viper (Calloselasma rhodostoma) in Southern Thailand: clinical and

laboratory correlations. Am. J. Trop. Med. Hyg. 35, 1235–1247 (1986).

18. Pierini, S. V, Warrell, D. A., De Paulo, A. & Theakston, R. D. G. High incidence of bites

and stings by snakes and other animals among rubber tappers and amazonian indians of the

Juruá Valley, Acre State, Brazil. Toxicon 34, 225–236 (1996).

19. Warrell, D. A. in Venomous Reptiles of the Western Hemisphere (eds. Campbell, J. R. &

Lamar, W. W.) 709–761 (Cornell University Press, 2004).

20. Myint-Lwin et al. Bites by Russell’s viper (Vipera russelli siamensis) in Burma:

haemostatic, vascular, and renal disturbances and response to treatment. Lancet 326, 1259–

1264 (1985).

21. Habib, A. G. et al. Envenoming after carpet viper (Echis ocellatus) bite during pregnancy:

timely use of effective antivenom improves maternal and foetal outcomes. Trop. Med. Int.

Heal. 13, 1172–1175 (2008).

49

22. Warrell, D. A. & Arnett, C. The importance of bites by the saw-scaled or carpet viper (Echis

carinatus): epidemiological studies in Nigeria and a review of the world literature. Acta

Trop. 33, 307–41 (1976).

23. Williams, D., Jensen, S., Nimorakiotakis, B. & Winkel, K. D. Venomous Bites and Stings in

Papua New Guinea (Australian Venom Research Unit, Parkville, 2005).

24. Warrell, D. A. & Ormerod, L. D. Snake venom ophthalmia and blindness caused by the

spitting cobra (Naja nigricollis ) in Nigeria. Am. J. Trop. Med. Hyg. 25, 525–529 (1976).

25. Smith, J. et al. Malignancy in chronic ulcers and scars of the leg (Marjolin’s ulcer): a study

of 21 patients. Skeletal Radiol. 30, 331–337 (2001).

26. Williams, S. S. et al. Delayed psychological morbidity associated with snakebite

envenoming. PLoS Negl. Trop. Dis. 5, e1255 (2011).

27. GBD 2013 DALYs and HALE Collaborators. Global, regional, and national disability-

adjusted life years (DALYs) for 306 diseases and injuries and healthy life expectancy

(HALE) for 188 countries, 1990-2013: quantifying the epidemiological transition. Lancet

386, 2145–91 (2015).

28. Fry, B. G. et al. The toxicogenomic multiverse: convergent recruitment of proteins into

animal venoms. Annu. Rev. Genomics Hum. Genet. 10, 483–511 (2009).

29. Daltry, J. C., Wüster, W. & Thorpe, R. S. Diet and snake venom evolution. Nature 379,

537–540 (1996).

30. Chippaux, J.-P., Williams, V. & White, J. Snake venom variability: methods of study, results

and interpretation. Toxicon 29, 1279–1303 (1991).

31. Olivera, B. M. Conus peptides: biodiversity-based discovery and exogenomics. J. Biol.

50

Chem. 281, 31173–31177 (2006).

32. Durban, J. et al. Integrated ‘omics’ profiling indicates that miRNAs are modulators of the

ontogenetic venom composition shift in the Central American rattlesnake, Crotalus simus

simus. BMC Genomics 14, 234 (2013).

33. Casewell, N. R. et al. Medically important differences in snake venom composition are

dictated by distinct postgenomic mechanisms. Proc. Natl. Acad. Sci. USA 111, 9205–9210

(2014).

34. Sanz, L. & Calvete, J. Insights into the evolution of a snake venom multi-gene family from

the genomic organization of Echis ocellatus SVMP genes. Toxins (Basel). 8, 216 (2016).

35. Dowell, N. L. et al. The deep origin and recent loss of venom toxin genes in rattlesnakes.

Curr. Biol. 26, 2434–2445 (2016).

36. Calvete, J. J. Venomics: integrative venom proteomics and beyond. Biochem. J. 474, 611–

634 (2017).

37. Reeks, T. A., Fry, B. G. & Alewood, P. F. Privileged frameworks from snake venom. Cell.

Mol. Life Sci. 72, 1939–1958 (2015).

38. Doley, R. & Kini, R. M. Protein complexes in snake venom. Cell. Mol. Life Sci. 66, 2851–

2871 (2009).

39. Vonk, F. J. et al. The king cobra genome reveals dynamic gene evolution and adaptation in

the snake venom system. Proc. Natl. Acad. Sci. U. S. A. 110, 20651–6 (2013).

40. Calvete, J. J. Snake venomics: From the inventory of toxins to biology. Toxicon 75, 44–62

(2013).

41. Rokyta, D. R., Wray, K. P., McGivern, J. J. & Margres, M. J. The transcriptomic and

51

proteomic basis for the evolution of a novel venom phenotype within the Timber Rattlesnake

(Crotalus horridus). Toxicon 98, 34–48 (2015).

42. Brahma, R. K., McCleary, R. J. R., Kini, R. M. & Doley, R. Venom gland transcriptomics

for identifying, cataloging, and characterizing venom proteins in snakes. Toxicon 93, 1–10

(2015).

43. Pla, D., Gutiérrez, J. M. & Calvete, J. J. Second generation antivenomics: comparing

immunoaffinity and immunodepletion protocols. Toxicon 60, 213–214 (2012).

44. Gutiérrez, J. M. et al. Assessing the preclinical efficacy of antivenoms: From the lethality

neutralization assay to antivenomics. Toxicon 69, 168–179 (2013).

45. Dixon, R. W. & Harris, J. B. Myotoxic Activity of the Toxic Phospholipase, Notexin, from

the Venom of the Australian Tiger Snake. J. Neuropathol. Exp. Neurol. 55, 1230–1237

(1996).

46. Montecucco, C., Gutiérrez, J. M. & Lomonte, B. Cellular pathology induced by snake

venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms

of action. Cell. Mol. Life Sci. 65, 2897–2912 (2008).

47. Gutiérrez, J. M. & Ownby, C. L. Skeletal muscle degeneration induced by venom

phospholipases A2: insights into the mechanisms of local and systemic myotoxicity. Toxicon

42, 915–931 (2003).

48. Gutiérrez, J. M., Rucavado, A., Chaves, F., Díaz, C. & Escalante, T. Experimental pathology

of local tissue damage induced by Bothrops asper snake venom. Toxicon 54, 958–975

(2009).

49. Hernández, R. et al. Poor regenerative outcome after skeletal muscle necrosis induced by

Bothrops asper venom: alterations in microvasculature and nerves. PLoS One 6, e19834

52

(2011).

50. Escalante, T., Rucavado, A., Fox, J. W. & Gutiérrez, J. M. Key events in microvascular

damage induced by snake venom hemorrhagic metalloproteinases. J. Proteomics 74, 1781–

1794 (2011).

51. Seo, T. et al. Haemorrhagic snake venom metalloproteases and human ADAMs cleave

LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo . FEBS

J. 284, 1657–1671 (2017).

52. Gutiérrez, J., Escalante, T., Rucavado, A., Herrera, C. & Fox, J. A Comprehensive View of

the Structural and Functional Alterations of Extracellular Matrix by Snake Venom

Metalloproteinases (SVMPs): Novel Perspectives on the Pathophysiology of Envenoming.

Toxins (Basel). 8, 304 (2016).

53. Jiménez, N., Escalante, T., Gutiérrez, J. M. & Rucavado, A. Skin Pathology Induced by

Snake Venom Metalloproteinase: Acute Damage, Revascularization, and Re-epithelization

in a Mouse Ear Model. J. Invest. Dermatol. 128, 2421–2428 (2008).

54. Rivel, M. et al. Pathogenesis of dermonecrosis induced by venom of the spitting cobra, Naja

nigricollis: an experimental study in mice. Toxicon 119, 171–179 (2016).

55. Dubovskii, P. V & Utkin, Y. N. Cobra cytotoxins: structural organization and antibacterial

activity. Acta Naturae 6, 11–8 (2014).

56. Mora, J., Mora, R., Lomonte, B. & Gutiérrez, J. M. Effects of Bothrops asper Snake Venom

on Lymphatic Vessels: Insights into a Hidden Aspect of Envenomation. PLoS Negl. Trop.

Dis. 2, e318 (2008).

57. Teixeira, C., Cury, Y., Moreira, V., Picolo, G. & Chaves, F. Inflammation induced by

Bothrops asper venom. Toxicon 54, 988–997 (2009).

53

58. Rucavado, A. et al. Viperid Envenomation Wound Exudate Contributes to Increased

Vascular Permeability via a DAMPs/TLR-4 Mediated Pathway. Toxins (Basel). 8, 349

(2016).

59. Zhang, C., Medzihradszky, K. F., Sánchez, E. E., Basbaum, A. I. & Julius, D. Lys49

myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release.

Proc. Natl. Acad. Sci. USA 114, E2524–E2532 (2017).

60. Barber, C. M., Isbister, G. K. & Hodgson, W. C. Alpha neurotoxins. Toxicon 66, 47–58

(2013).

61. Rossetto, O. & Montecucco, C. Presynaptic Neurotoxins with Enzymatic Activities.

Handbook of Experimental Pharmacology 184, 129–170 (2008). doi:10.1007/978-3-540-

74805-2_6

62. Pungerčar, J. & Križaj, I. Understanding the molecular mechanism underlying the

presynaptic toxicity of secreted phospholipases A2. Toxicon 50, 871–892 (2007).

63. Paoli, M. et al. Mass spectrometry analysis of the phospholipase A2activity of snake pre-

synaptic neurotoxins in cultured neurons. J. Neurochem. 111, 737–744 (2009).

64. Harris, J. B., Grubb, B. D., Maltin, C. A. & Dixon, R. The Neurotoxicity of the Venom

Phospholipases A2, Notexin and Taipoxin. Exp. Neurol. 161, 517–526 (2000).

65. Prasarnpun, S., Walsh, J. & Harris, J. B. β-bungarotoxin-induced depletion of synaptic

vesicles at the mammalian neuromuscular junction. Neuropharmacology 47, 304–314

(2004).

66. Rigoni, M. et al. Snake Phospholipase A2Neurotoxins Enter Neurons, Bind Specifically to

Mitochondria, and Open Their Transition Pores. J. Biol. Chem. 283, 34013–34020 (2008).

54

67. Harvey, A. & Robertson, B. Dendrotoxins: Structure-Activity Relationships and Effects on

Potassium Ion Channels. Curr. Med. Chem. 11, 3065–3072 (2004).

68. Harvey, A. L. in Handbook of Venoms and Toxins of Reptiles (ed. Mackessy, S. P.) 317–

324 (CRC Press, Boca Raton, 2010).

69. Fox, J. W. & Serrano, S. M. T. Structural considerations of the snake venom

metalloproteinases, key members of the M12 reprolysin family of metalloproteinases.

Toxicon 45, 969–985 (2005).

70. White, J. Snake venoms and coagulopathy. Toxicon 45, 951–967 (2005).

71. Del Brutto, O. H. & Del Brutto, V. J. Neurological complications of venomous snake bites: a

review. Acta Neurol. Scand. 125, 363–372 (2011).

72. Kini, R. M. The intriguing world of prothrombin activators from snake venom. Toxicon 45,

1133–1145 (2005).

73. Kini, R. & Koh, C. Metalloproteases Affecting Blood Coagulation, Fibrinolysis and Platelet

Aggregation from Snake Venoms: Definition and Nomenclature of Interaction Sites. Toxins

(Basel). 8, 284 (2016).

74. Yamashita, K. M., Alves, A. F., Barbaro, K. C. & Santoro, M. L. Bothrops jararaca venom

metalloproteinases are essential for coagulopathy and increase plasma tissue factor levels

during envenomation. PLoS Negl. Trop. Dis. 8, e2814 (2014).

75. Rucavado, A. et al. Thrombocytopenia and platelet hypoaggregation induced by Bothrops

asper snake venom: toxins involved and their contribution to metalloproteinase-induced

pulmonary hemorrhage. Thromb. Haemost. 94, 123-131 (2005). doi:10.1160/th05-02-0112

76. Calvete, J. J. et al. Snake venom disintegrins: evolution of structure and function. Toxicon

55

45, 1063–1074 (2005).

77. Du, X. Y. & Clemetson, K. J. in Handbook of Venoms and Toxins of Reptiles (ed. Mackessy,

S. P.) 359–375 (CRC Press, Boca Raton, 2010).

78. Resiere, D., Mégarbane, B., Valentino, R., Mehdaoui, H. & Thomas, L. Bothrops

lanceolatus bites: guidelines for severity assessment and emergent management. Toxins

(Basel). 2, 163–173 (2010).

79. Than-Than et al. Contribution of focal haemorrhage and microvascular fibrin deposition to

fatal envenoming by Russell’s viper (Vipera russelli siamensis) in Burma. Acta Trop. 46,

23–38 (1989).

80. Hayashi, M. A. F. & Camargo, A. C. M. The Bradykinin-potentiating peptides from venom

gland and brain of Bothrops jararaca contain highly site specific inhibitors of the somatic

angiotensin-converting enzyme. Toxicon 45, 1163–1170 (2005).

81. Höjer, J., Tran Hung, H. & Warrell, D. Life-threatening hyponatremia after krait bite

envenoming – A new syndrome. Clin. Toxicol. 48, 956–957 (2010).

82. Sitprija, V. & Sitprija, S. Renal effects and injury induced by animal toxins. Toxicon 60,

943–953 (2012).

83. Pinho, F. M. O., Zanetta, D. M. T. & Burdmann, E. A. Acute renal failure after Crotalus

durissus snakebite: A prospective survey on 100 patients. Kidney Int. 67, 659–667 (2005).

84. Warrell, D. A. in Venomous snakes. Ecology, Evolution and Snakebite (eds. Thorpe, R. S.,

Wuster, W. & Malhotra, A.) 189–203 (Clarendon Press, 1997).

85. Harris, J. B. et al. Snake bite in Chittagong Division, Bangladesh: a study of bitten patients

who developed no signs of systemic envenoming. Trans. R. Soc. Trop. Med. Hyg. 104, 320–

56

327 (2010).

86. WHO Regional Office for Africa. Guidelines for the Prevention and Clinical Management of

Snakebite in Africa [online], http://www.afro.who.int/pt/grupos-organicos-e-

programas/2731-guidelines-for-the-prevention-and-clinical-management-of-snakebite-in-

africa.html (2010).

87. WHO Regional Office for South-East Asia. Guidelines for the Management of Snakebites

[online], http://apps.searo.who.int/PDS_DOCS/B5255.pdf?ua=1 (2016).

88. Sutherland, S. K. & Tibballs, J. Australian Animal Toxins: The Creatures, their Toxins and

Care of the Poisoned Patient, 2nd Ed. (Oxford University Press, Melbourne, 2001). 89.

White, J. A Clinician’s Guide to Australian Venomous Bites and Stings (bioCSL, Parkville,

2013).

90. Weinstein, S., Warrell, D., White, J. & Keyler, D. ‘Venomous’ Bites from non-Venomous

Snakes: A Critical Analysis of Risk and Management of ‘Colubrid’ Snake Bites (Elsevier,

Waltham, Massachusettes, 2011).

91. Weinstein, S. A., White, J., Keyler, D. E. & Warrell, D. A. Non-front-fanged colubroid

snakes: A current evidence-based analysis of medical significance. Toxicon 69, 103–113

(2013).

92. Warrell, D. A. et al. Severe neurotoxic envenoming by the Malayan krait Bungarus candidus

(Linnaeus): response to antivenom and anticholinesterase. BMJ 286, 678–680 (1983).

93. Ariaratnam, C. A., Sheriff, M. H. R., Theakston, R. D. G. & Warrell, D. A. Distinctive

epidemiologic and clinical features of common krait (Bungarus caeruleus) bites in Sri

Lanka. Am. J. Trop. Med. Hyg. 79, 458–62 (2008).

94. Russell, F. E. Snake venom poisoning. (JB Lippincott, 1980).

57

95. Ariaratnam, C. A., Sheriff, M. H. R., Arambepola, C., Theakston, R. D. G. & Warrell, D. A.

Syndromic Approach to Treatment of Snake Bite in Sri Lanka Based on Results of a

Prospective National Hospital-Based Survey of Patients Envenomed by Identified Snakes.

Am. J. Trop. Med. Hyg. 81, 725–731 (2009).

96. Warrell, D. A. et al. Poisoning by bites of the saw-scaled or carpet viper (Echis carinatus) in

Nigeria. QJ Med. 46, 33–62 (1977).

97. Sano-Martins, I. S. et al. Reliability of the simple 20 minute whole blood clotting test

(WBCT20) as an indicator of low plasma fibrinogen concentration in patients envenomed by

Bothrops snakes. Toxicon 32, 1045–1050 (1994).

98. Wood, D., Sartorius, B. & Hift, R. Ultrasound findings in 42 patients with cytotoxic tissue

damage following bites by South African snakes. Emerg. Med. J. 33, 477–481 (2016).

99. Theakston, R. & Laing, G. Diagnosis of Snakebite and the Importance of Immunological

Tests in Venom Research. Toxins (Basel). 6, 1667–1695 (2014).

100. Ho, M., Warrell, M. J., Warrell, D. A., Bidwell, D. & Voller, A. A critical reappraisal of the

use of enzyme-linked immunosorbent assays in the study of snake bite. Toxicon 24, 211–221

(1986).

101. Dong, L. Immunogenicity of venoms from four common snakes in the South of Vietnam and

development of ELISA kit for venom detection. J. Immunol. Methods 282, 13–31 (2003).

102. Kulawickrama, S. et al. Development of a sensitive enzyme immunoassay for measuring

taipan venom in serum. Toxicon 55, 1510–1518 (2010).

103. Sutherland, S. K. Rapid venom identification: availability of kits. Med. J. Aust. 2, 602–3

(1979).

58

104. Chen, T. et al. Unmasking venom gland transcriptomes in reptile venoms. Anal. Biochem.

311, 152–156 (2002).

105. Sharma, S. K. et al. Use of Molecular Diagnostic Tools for the Identification of Species

Responsible for Snakebite in Nepal: A Pilot Study. PLoS Negl. Trop. Dis. 10, e0004620

(2016).

106. Tun-Pe et al. Acceptability study of protective boots among farmers of Taungdwingyi

Township in Management of Snakebite and Research (7-11) [online],

http://apps.searo.who.int/PDS_DOCS/B3205.pdf (2002).

107. Chappuis, F., Sharma, S. K., Jha, N., Loutan, L. & Bovier, P. A. Protection against snake

bites by sleeping under a bed net in southeastern Nepal. Am. J. Trop. Med. Hyg. 77, 197–9

(2007).

108. Sharma, S. K. et al. Effectiveness of Rapid Transport of Victims and Community Health

Education on Snake Bite Fatalities in Rural Nepal. Am. J. Trop. Med. Hyg. 89, 145–150

(2013).

109. Tun-Pe, Aye-Aye-Myint, Khin-Ei-Han, Thi-Ha & Tin-Nu-Swe. Local compression pads as a

first-aid measure for victims of bites by Russell’s viper (Daboia russelii siamensis) in

Myanmar. Trans. R. Soc. Trop. Med. Hyg. 89, 293–295 (1995).

110. Avau, B., Borra, V., Vandekerckhove, P. & De Buck, E. The Treatment of Snake Bites in a

First Aid Setting: A Systematic Review. PLoS Negl. Trop. Dis. 10, e0005079 (2016).

111. Faiz, M. A. et al. Bites by the Monocled Cobra, Naja kaouthia, in Chittagong Division,

Bangladesh: Epidemiology, Clinical Features of Envenoming and Management of 70

Identified Cases. Am. J. Trop. Med. Hyg. 16–842 (2017). doi:10.4269/ajtmh.16-0842

112. Watt, G., Theakston, R. D. G., Padre, L., Laughlin, L. W. & Tuazon, M. L. Tourniquet

59

Application after Cobra Bite: Delay in the Onset of Neurotoxicity and the Dangers of

Sudden Release. Am. J. Trop. Med. Hyg. 38, 618–622 (1988).

113. Audebert, F., Sorkine, M. & Bon, C. Envenoming by viper bites in France: Clinical

gradation and biological quantification by ELISA. Toxicon 30, 599–609 (1992).

114. Bucher, B. et al. Clinical indicators of envenoming and serum levels of venom antigens in

patients bitten by Bothrops lanceolatusin Martinique. Trans. R. Soc. Trop. Med. Hyg. 91,

186–190 (1997).

115. WHO. Guidelines for the Production, Control and Regulation of Snake Antivenom

Immunoglobulins [online], http://www.who.int/bloodproducts/snake_antivenoms/en (2010).

116. Gutiérrez, J. M., León, G. & Lomonte, B. Pharmacokinetic-Pharmacodynamic Relationships

of Immunoglobulin Therapy for Envenomation. Clin. Pharmacokinet. 42, 721–741 (2003).

117. Habib, A. G. & Warrell, D. A. Antivenom therapy of carpet viper (Echis ocellatus)

envenoming: effectiveness and strategies for delivery in West Africa. Toxicon 69, 82–89

(2013).

118. Theakston, R. D. G. & Warrell, D. A. Crisis in snake antivenom supply for Africa. Lancet

356, 2104 (2000).

119. Warrell, D. A. et al. Bites by the saw-scaled or carpet viper (Echis carinatus): trial of two

specific antivenoms. Br. Med. J. 4, 437–40 (1974).

120. Cardoso, J.L. et al. Randomized comparative trial of three antivenoms in the treatment of

envenoming by lance-headed vipers (Bothrops jararaca) in São Paulo, Brazil. Q. J. Med 86,

315-325 (1993).

121. Smalligan, R. Crotaline snake bite in the Ecuadorian Amazon: randomised double blind

Con formato: Inglés (Estados Unidos)

60

comparative trial of three South American polyspecific antivenoms. BMJ 329, 1120–1129

(2004).

122. Abubakar, S. B. et al. Pre-clinical and preliminary dose-finding and safety studies to identify

candidate antivenoms for treatment of envenoming by saw-scaled or carpet vipers (Echis

ocellatus) in northern Nigeria. Toxicon 55, 719–723 (2010).

123. Abubakar, I. S. et al. Randomised Controlled Double-Blind Non-Inferiority Trial of Two

Antivenoms for Saw-Scaled or Carpet Viper (Echis ocellatus) Envenoming in Nigeria. PLoS

Negl. Trop. Dis. 4, e767 (2010).

124. de Silva, H. A. et al. Low-Dose Adrenaline, Promethazine, and Hydrocortisone in the

Prevention of Acute Adverse Reactions to Antivenom following Snakebite: A Randomised,

Double-Blind, Placebo-Controlled Trial. PLoS Med. 8, e1000435 (2011).

125. Watt, G. et al. Positive Response to Edrophonium in Patients with Neurotoxic Envenoming

by Cobras (Naja naja philippinensis). N. Engl. J. Med. 315, 1444–1448 (1986).

126. Golnik, K. C., Pena, R., Lee, A. G. & Eggenberger, E. R. An ice test for the diagnosis of

myasthenia gravis. Ophthalmology 106, 1282–1286 (1999).

127. Jorge, M. T. et al. Failure of chloramphenicol prophylaxis to reduce the frequency of

abscess formation as a complication of envenoming by Bothrops snakes in Brazil: a double-

blind randomized controlled trial. Trans. R. Soc. Trop. Med. Hyg. 98, 529–534 (2004).

128. Darracq, M. A., Cantrell, F. L., Klauk, B. & Thornton, S. L. A chance to cut is not always a

chance to cure- fasciotomy in the treatment of rattlesnake envenomation: A retrospective

poison center study. Toxicon 101, 23–26 (2015).

129. Jayawardana, S., Gnanathasan, A., Arambepola, C. & Chang, T. Chronic Musculoskeletal

Disabilities following Snake Envenoming in Sri Lanka: A Population-Based Study. PLoS

61

Negl. Trop. Dis. 10, e0005103 (2016).

130. Hasan, S. M. K., Basher, A., Molla, A. A., Sultana, N. K. & Faiz, M. A. The impact of snake

bite on household economy in Bangladesh. Trop. Doct. 42, 41–43 (2012).

131. Vaiyapuri, S. et al. Snakebite and Its Socio-Economic Impact on the Rural Population of

Tamil Nadu, India. PLoS One 8, e80090 (2013).

132. Aye, K.-P. et al. Clinical and laboratory parameters associated with acute kidney injury in

patients with snakebite envenomation: a prospective observational study from Myanmar.

BMC Nephrol. 18, 92 (2017).

133. Krishnamurthy, S., Gunasekaran, K., Mahadevan, S., Bobby, Z. & Kumar, A. P. Russell’s

viper envenomation-associated acute kidney injury in children in Southern India. Indian

Pediatr. 52, 583–586 (2015).

134. Muhammed, A. et al. Predictors of depression among patients receiving treatment for

snakebite in General Hospital, Kaltungo, Gombe State, Nigeria: August 2015. Int. J. Ment.

Health Syst. 11, (2017).

135. Pawade, B. S. et al. Rapid and selective detection of experimental snake envenomation –

Use of gold nanoparticle based lateral flow assay. Toxicon 119, 299–306 (2016).

136. Maduwage, K., O’Leary, M. A. & Isbister, G. K. Diagnosis of snake envenomation using a

simple phospholipase A2 assay. Sci. Rep. 4, (2014).

137. Harrison, R. A. et al. Research strategies to improve snakebite treatment: Challenges and

progress. J. Proteomics 74, 1768–1780 (2011).

138. Harrison, R. & Gutiérrez, J. Priority Actions and Progress to Substantially and Sustainably

Reduce the Mortality, Morbidity and Socioeconomic Burden of Tropical Snakebite. Toxins

62

(Basel). 8, 351 (2016).

139. Wagstaff, S. C., Laing, G. D., Theakston, R. D. G., Papaspyridis, C. & Harrison, R. A.

Bioinformatics and Multiepitope DNA Immunization to Design Rational Snake Antivenom.

PLoS Med. 3, e184 (2006).

140. Casewell, N. R., Wüster, W., Vonk, F. J., Harrison, R. A. & Fry, B. G. Complex cocktails:

the evolutionary novelty of venoms. Trends Ecol. Evol. 28, 219–229 (2013).

141. Ramos, H. R. et al. A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost

Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus

(Coral Snake) Venom. PLoS Negl. Trop. Dis. 10, e0004484 (2016).

142. Laustsen, A. et al. From Fangs to Pharmacology: The Future of Snakebite Envenoming

Therapy. Curr. Pharm. Des. 22, 5270–5293 (2016).

143. Engmark, M. et al. High-throughput immuno-profiling of mamba (Dendroaspis) venom

toxin epitopes using high-density peptide microarrays. Sci. Rep. 6, (2016).

144. Laustsen, A. H., Johansen, K. H., Engmark, M. & Andersen, M. R. Recombinant snakebite

antivenoms: A cost-competitive solution to a neglected tropical disease? PLoS Negl. Trop.

Dis. 11, e0005361 (2017).

145. Lewin, M., Samuel, S., Merkel, J. & Bickler, P. Varespladib (LY315920) Appears to Be a

Potent, Broad-Spectrum, Inhibitor of Snake Venom Phospholipase A2 and a Possible Pre-

Referral Treatment for Envenomation. Toxins (Basel). 8, 248 (2016).

146. O’Brien, J., Lee, S.-H., Onogi, S. & Shea, K. J. Engineering the Protein Corona of a

Synthetic Polymer Nanoparticle for Broad-Spectrum Sequestration and Neutralization of

Venomous Biomacromolecules. J. Am. Chem. Soc. 138, 16604–16607 (2016).

63

147. Saul, M. E. et al. A pharmacological approach to first aid treatment for snakebite. Nat. Med.

17, 809–811 (2011).

148. Rucavado, A. et al. Inhibition of local hemorrhage and dermonecrosis induced by Bothrops

asper snake venom: effectiveness of early in situ administration of the peptidomimetic

metalloproteinase inhibitor batimastat and the chelating agent CaNa2EDTA. Am. J. Trop.

Med. Hyg. 63, 313–319 (2000).

149. Azofeifa, K., Angulo, Y. & Lomonte, B. Ability of fucoidan to prevent muscle necrosis

induced by snake venom myotoxins: Comparison of high- and low-molecular weight

fractions. Toxicon 51, 373–380 (2008).

150. Prado, N. D. R. et al. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom

and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

PLoS One 11, e0151363 (2016).

151. Richard, G. et al. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-

Domain Antibodies (VHHs) and a VHH-Fc Antibody. PLoS One 8, e69495 (2013).

152. Gutiérrez, J. M. Improving antivenom availability and accessibility: Science, technology,

and beyond. Toxicon 60, 676–687 (2012).

153. Lancet, T. Snake bite—the neglected tropical disease. Lancet 386, 1110 (2015).

154. King, G.F. (editor) Venoms to Drugs: Venom as a Source for the Development of Human

Therapeutics (Royal Society of Chemistry Publishing, Cambridge, UK, 2015).

155. Eichberg, S., Sanz, L., Calvete, J. J. & Pla, D. Constructing comprehensive venom proteome

reference maps for integrative venomics. Expert Rev. Proteomics 12, 557–573 (2015).

156. Petras, D., Heiss, P., Süssmuth, R. D. & Calvete, J. J. Venom Proteomics of Indonesian King

64

Cobra,Ophiophagus hannah: Integrating Top-Down and Bottom-Up Approaches. J.

Proteome Res. 14, 2539–2556 (2015).

157. Petras, D., Heiss, P., Harrison, R. A., Süssmuth, R. D. & Calvete, J. J. Top-down venomics

of the East African green mamba, Dendroaspis angusticeps , and the black mamba,

Dendroaspis polylepis, highlight the complexity of their toxin arsenals. J. Proteomics 146,

148–164 (2016).

158. Catherman, A. D., Skinner, O. S. & Kelleher, N. L. Top Down proteomics: Facts and

perspectives. Biochem. Biophys. Res. Commun. 445, 683–693 (2014).

159. Fornelli, L. et al. Advancing Top-down Analysis of the Human Proteome Using a Benchtop

Quadrupole-Orbitrap Mass Spectrometer. J. Proteome Res. 16, 609–618 (2016).

160. Calderón-Celis, F. et al. Elemental Mass Spectrometry for Absolute Intact Protein

Quantification without Protein-Specific Standards: Application to Snake Venomics. Anal.

Chem. 88, 9699–9706 (2016).